TY - JOUR AB - Hepatitis B virus (HBV) infection is a significant global health threat, resulting in more than 800,000 deaths annually. Since HBV naturally infects only humans and chimpanzees, the development and evaluation of new therapies for chronic HBV infection are hindered by the lack of suitable animal models. Human sodium-taurocholate cotransporting polypeptide (NTCP) is a critical factor for HBV binding and entry, exhibiting species-specific differences in the amino acid sequences. This study investigated NTCP orthologs from various species to determine their capability to support HBV binding and infection. We demonstrate that nonhuman NTCP orthologs from woodchuck, ferret, aardvark, horse, rabbit, whale, big brown bat, cat, and rhinoceros support HBV binding and cellular entry, thereby rendering HepG2 cells susceptible to HBV infection upon expression. NTCP orthologs from hamster, goat, and cow support HBV binding but require specific amino acid exchanges to facilitate HBV infection. We show that replacement of the functional region, amino acids (aa) 84-87, in hamster NTCP with the human counterpart allows infection of HepG2 cells expressing the chimeric NTCP variant. Furthermore, we demonstrate that aa 82 in goat and cow NTCP, close to this functional region, needs to be modified to support HBV infection. This study could help identify previously unknown HBV reservoirs and may facilitate the establishment of new animal models.IMPORTANCEThe bona fide HBV entry receptor NTCP provides a natural barrier for cross-species transmission. We identified species-specific NTCP orthologues from woodchuck, ferret, aardvark, horse, rabbit, whale, big brown bat, cat, and rhinoceros that support HBV infection. This may reveal potential HBV reservoirs and facilitate the development of new HBV animal models. AU - Chen, F. AU - Wettengel, J.M. AU - Gegenfurtner, F. AU - Moosmüller, J. AU - Bunse, T. AU - Jeske, S. AU - Hagen, P. AU - Ni, Y.* AU - Urban, S.* AU - Protzer, U. C1 - 73588 C2 - 57120 TI - Identification of NTCP animal orthologs supporting hepatitis B virus binding and infection. JO - J. Virol. VL - 99 IS - 4 PY - 2025 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) is usually maintained latently on passaging in cell culture, as are most herpesviruses in vivo. Its fitness, or its ability to infect and replicate in naïve cells, cannot be ascertained by serially passaging it in host cells because no identified cell line initially supports a productive infection by it. Yet its fitness is critical to EBV's remarkable success as a human pathogen. We have, therefore, developed multiple approaches to assess EBV's fitness upon being reactivated from its familiar state of latency. We established and tested expression plasmids for 77 viral genes and a set of shRNAs targeting 25 viral genes to measure how increasing and decreasing their levels affected the fitness of the released stocks of virus. Four of their properties were then analyzed: (i) their concentrations of physical particles, (ii) their binding to the CD21 receptor on a B-cell line, (iii) their entry into human primary B cells, and (iv) their infectious titers. These analyses identified multiple EBV genes whose altered levels of expression altered the biological activities of the released virus. These measurements revealed, though, an unexpected insight into the robustness of EBV produced from latently infected cells. EBV is amazingly resilient to any increased expression of its genes. The levels expressed in cells, as they support an induced productive infection, therefore are close to optimal for the fitness of the released virus.IMPORTANCEPopulations of viruses accumulate mutations while being propagated. While most mutations are neutral or disadvantageous, some confer on the variant a selective advantage, increasing its infectivity. These variants can be identified by serial passaging virus stocks, allowing those with increased fitness to predominate. This approach does not work for Epstein-Barr virus (EBV), for which no identified cell line initially supports its productive infection. How mutations accumulate in EBV as it is propagated latently to affect its fitness was unknown. We have devised an approach to assess EBV's fitness upon being reactivated. Our findings suggest that EBV during its many latent generations has maintained a strikingly robust productive fitness. AU - Chen, Y.-F. A. AU - Mocanu, B. AU - Akidil, E. AU - Pich, D. AU - Mautner, J. AU - Sugden, B.* AU - Hammerschmidt, W. C1 - 74839 C2 - 57626 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Assessing the fitness of Epstein-Barr virus following its reactivation. JO - J. Virol. VL - 99 IS - 7 PB - Amer Soc Microbiology PY - 2025 SN - 0022-538X ER - TY - JOUR AB - Extracellular vesicles (EVs) are released from all cells of the body. They are considered to mirror the state of the cells from which they are released and circulate in the blood, suggesting a possible use of EV analysis for diagnostic purposes. Here, we report that the analysis of single EVs by flow cytometry can detect infection of cells with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying expression of the SARS-CoV-2 spike (S) protein on the surface of EVs and the cellular origin of EVs by detecting cell-type-specific markers such as troponin (cTNT1) for cardiomyocytes. In coronavirus-associated disease 19 (COVID-19) patients, we detected a direct correlation of the frequencies of circulating S-expressing EVs, but not of cTNT/S-co-expressing EVs, with the subsequent development of a severe disease course. Detection of circulating S-expressing EVs indicates widespread SARS-CoV-2 infection in the body, which may contribute to the immune pathogenesis that triggers tissue and organ damage in COVID-19. Our findings suggest that detecting circulating viral antigen-expressing EVs may provide crucial predictive information on infection-associated disease courses in situations of a future viral pandemic.IMPORTANCEThe ability to predict which patients infected with the SARS-CoV-2 virus will develop severe disease remains a significant clinical challenge. The present study demonstrates that EVs in the peripheral blood, carrying the SARS-CoV-2 spike protein, can be detected by flow cytometry and serve as early biomarkers of disease progression. In contradistinction to PCR or serology, this method provides insight into systemic viral spread and potential organ involvement. The early identification of spike-positive EVs at the time of hospital admission has the potential to facilitate the timely identification of high-risk patients, thereby enhancing the efficacy of triage and subsequent care. This approach may also be of value in terms of facilitating a more rapid and precise response to future virus pandemics. AU - Hammer, E.* AU - Flynn, C.* AU - Rössler, J. AU - Erder, J.* AU - Napieralski, R.* AU - Fricke, L.* AU - Campbell, B.* AU - Feuerherd, M. AU - Esslinger, F. AU - von Brunn, A. * AU - Weber, T.* AU - King, S.* AU - Tan, S.* AU - Brisson, A.R.* AU - Protzer, U. AU - Schricker, G.* AU - Gärtner, K.* AU - Ebert, G. AU - Moretti, A.* AU - Klein, F.* AU - Knoops, K.* AU - Heeren, R.* AU - Hammerschmidt, W. AU - Zeidler, R. AU - Wilhelm, O.* AU - Knolle, P.A.* AU - Höchst, B.* C1 - 75611 C2 - 58243 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Prediction of COVID-19 disease progression by multiparametric analysis of circulating extracellular vesicles with flow cytometry. JO - J. Virol. VL - 99 IS - 10 PB - Amer Soc Microbiology PY - 2025 SN - 0022-538X ER - TY - JOUR AB - Class II genotype IV Newcastle disease virus (NDV), a historically virulent strain responsible for the first Newcastle disease (ND) panzootic from the 1940s to 1960s, was presumed extinct after its last reported isolate in India in 2000. Here, we report the emergence of four virulent genotype IV NDV isolates from 6,731 wild birds and domestic poultry across nine provinces of China between 2021 and 2023, representing the first genetically confirmed isolation of this ancestral genotype in over two decades. These isolates exhibit remarkably high genetic similarity to ancestral strains, showing minimal divergence despite a temporal span of 50-90 years, yet they differ from the most recently reported isolate from India. Infection with the representative isolate, KS02, caused severe lethality and higher transmissibility in specific-pathogen-free chicks than a genotype VII reference virus. Notably, LaSota vaccination provided only limited protection at the conventional hemagglutination inhibition (HI) threshold and achieved complete protection only when HI titers were at least twofold higher, in contrast to the protection observed against genotype VII challenge. This unusual genetic stability raises concerns about the origin and evolutionary history of these viruses as well as highlights the urgent need to update vaccination strategies, including approaches to elicit higher HI titers through intensified immunization schedules. This study underscores the critical importance of sustained global surveillance of genotype IV NDV in domestic poultry and migratory birds to monitor its spread and evolution.IMPORTANCEVirulent Newcastle disease virus (NDV), particularly emerging isolates, poses a major threat to poultry health and production, causing severe morbidity, high mortality, and economic losses. While ancestral class II genotypes II and IX have persisted globally across various bird species, the status of genotype IV NDV-last reported in India in 2000-had been uncertain. This study documents the emergence of genotype IV isolates in wild and domestic birds across China from 2021 to 2023, marking their return after more than two decades of presumed extinction. The representative isolate, KS02, showed severe lethality and high transmissibility in chicks compared to the circulating virus. The LaSota vaccine conferred complete protection against this isolate only when HI titers were at least twofold above the conventional protective threshold. These findings underscore the significant risk posed by reemerging genotype IV NDV, highlighting the urgent need for surveillance and updated vaccination strategies. AU - Yan, W.* AU - Liu, X.* AU - Jiang, S.* AU - Li, H.* AU - Chi, W.* AU - Luo, R.* AU - Peng, J.* AU - Jiang, F.* AU - Stöger, T. AU - Wajid, A.* AU - Dodovski, A.* AU - Gao, C.* AU - Mingala, C.N.* AU - Andreychuk, D.B.* AU - Yin, R.* C1 - 75993 C2 - 58335 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Emergence and characterization of historically extinct virulent genotype IV Newcastle disease virus in wild and domestic birds: genetic insights, pathogenicity, and vaccine efficacy. JO - J. Virol. PB - Amer Soc Microbiology PY - 2025 SN - 0022-538X ER - TY - JOUR AB - Cellular immunotherapy is a proven approach against Epstein-Barr virus (EBV)-driven lymphoproliferation in recipients of hematopoietic stem cells. Extending the applicability and improving the response rates of such therapy demands improving the knowledge base. We studied 23 healthy donors for specific CD4(+) T cell responses against the viral tegument protein BNRF1 and found such T cells in all seropositive donors, establishing BNRF1 as an important immune target in EBV. We identified 18 novel immune epitopes from BNRF1, all of them generated by natural processing of the full-length protein from virus-transformed lymphoblastoid cell lines (LCL). BNRF1-specific CD4(+) T cells were measured directly ex vivo by a cytokine-based method, thus providing a tool to study the interaction between immunity and infection in health and disease. T cells of the cytotoxic Th1 type inhibited the proliferation of autologous LCL as well as virus-driven transformation. We infer that they are important in limiting reactivations to subclinical levels during health and reducing virus propagation during disease. The information obtained from this work will feed into data sets that are indispensable in the design of patient-tailored immunotherapeutic approaches, thereby enabling the stride toward broader application of T cell therapy and improving clinical response rates.IMPORTANCE Epstein-Barr virus is carried by most humans and can cause life-threatening diseases. Virus-specific T cells have been used in different clinical settings with variable success rates. One way to improve immunotherapy is to better suit T cell generation protocols to viral targets available in different diseases. BNRF1 is present in viral particles and therefore likely available as a target for T cells in diseases with virus amplification. Here, we studied healthy Epstein-Barr virus (EBV) carriers for BNRF1 immunogenicity and report our results indicating BNRF1 to be a dominant target of the EBV-specific CD4(+) T cell response. BNRF1-specific CD4(+) T cells were found to be cytotoxic and capable of limiting EBV-driven B cell transformation in vitro. The findings of this work contribute to forwarding our understanding of host-virus interactions during health and disease and are expected to find direct application in the generation of specific T cells for immunotherapy. AU - Adhikary, D. AU - Damaschke, J. AU - Mautner, J. AU - Behrends, U. C1 - 59236 C2 - 48682 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - The Epstein-Barr virus major tegument protein BNRF1 is a common target of cytotoxic CD4+ T cells. JO - J. Virol. VL - 94 IS - 15 PB - Amer Soc Microbiology PY - 2020 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus (EBV) causes human cancers, and epidemiological studies have shown that lytic replication is a risk factor for some of these tumors. This fits with the observation that EBV M81, which was isolated from a Chinese patient with nasopharyngeal carcinoma, induces potent virus production and increases the risk of genetic instability in infected B cells. To find out whether this property extends to viruses found in other parts of the world, we investigated 22 viruses isolated from Western patients. While one-third of the viruses hardly replicated, the remaining viruses showed variable levels of replication, with three isolates replicating at levels close to that of M81 in B cells. We cloned one strongly replicating virus into a bacterial artificial chromosome (BAC); the resulting recombinant virus (MSHJ) retained the properties of its nonrecombinant counterpart and showed similarities to M81, undergoing lytic replication in vitro and in vivo after 3 weeks of latency. In contrast, B cells infected with the nonreplicating Western B95-8 virus showed early but abortive replication accompanied by cytoplasmic BZLF1 expression. Sequencing confirmed that rMSHJ is a Western virus, being genetically much closer to 695-8 than to M81. Spontaneous replication in rM81- and rMSHJ-infected B cells was dependent on phosphorylated Btk and was inhibited by exposure to ibrutinib, opening the way to clinical intervention in patients with abnormal EBV replication. As rMSHJ contains the complete EBV genome and induces lytic replication in infected B cells, it is ideal to perform genetic analyses of all viral functions in Western strains and their associated diseases.IMPORTANCE The Epstein-Barr virus (EBV) infects the majority of the world population but causes different diseases in different countries. Evidence that lytic replication, the process that leads to new virus progeny, is linked to cancer development is accumulating. Indeed, viruses such as M81 that were isolated from Far Eastern nasopharyngeal carcinomas replicate strongly in B cells. We show here that some viruses isolated from Western patients, including the MSHJ strain, share this property. Moreover, replication of both M81 and of MSHJ was sensitive to ibrutinib, a commonly used drug, thereby opening an opportunity for therapeutic intervention. Sequencing of MSHJ showed that this virus is quite distant from M81 and is much closer to nonreplicating Western viruses. We conclude that Western EBV strains are heterogeneous, with some viruses being able to replicate more strongly and therefore being potentially more pathogenic than others, and that the virus sequence information alone cannot predict this property. AU - Delecluse, S.* AU - Poirey, R.* AU - Zeier, M.* AU - Schnitzler, P.* AU - Behrends, U. AU - Tsai, M.H.* AU - Delecluse, H.J.* C1 - 58488 C2 - 48229 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Identification and cloning of a new western Epstein-Barr virus strain that replicates efficiently in primary B cells. JO - J. Virol. VL - 94 IS - 10 PB - Amer Soc Microbiology PY - 2020 SN - 0022-538X ER - TY - JOUR AB - Murine gammaherpesvirus 68 (MHV68) is a small-animal model suitable for study of the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal Toll-like receptor (TLR) escort protein UNC93B, endosomal TLR7, -9, and -13, and cell surface TLR2 in MHV68 detection. We found that the alpha interferon (IFN-alpha) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9(-/-) cells compared to levels in wild type (WT) cells but not completely lost. Tlr7(-/-) pDC responded similarly to WT. However, we found that in Unc93b(-/-) pDC, as well as in Tlr7(-/-) Tlr9(-/-) double-knockout pDC, the IFN-alpha response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFN-alpha response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7(-/-) Tlr9(-/-) mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b(-/-), Tlr7(-/-), Tlr9(-/-), Tlr7(-/-), and Tlr9(-/-) splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7(-/-) Tlr9(-/-) splenocytes compared to levels in the WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.IMPORTANCE The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific, and therefore the homolog murine gammaherpesvirus 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here, we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as on the control of this virus in its host. AU - Bussey, K.A.* AU - Murthy, S.* AU - Reimer, E.* AU - Chan, B.* AU - Hatesuer, B.* AU - Schughart, K.* AU - Glaunsinger, B.* AU - Adler, H. AU - Brinkmann, M.M.* C1 - 54752 C2 - 45789 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - e01173-18 TI - Endosomal toll-like receptors 7 and 9 cooperate in detection of murine gammaherpesvirus 68 infection. JO - J. Virol. VL - 93 IS - 3 PB - Amer Soc Microbiology PY - 2019 SN - 0022-538X ER - TY - JOUR AB - With a yearly death toll of 880,000, hepatitis B virus (HBV) remains a major health problem worldwide, despite an effective prophylactic vaccine and well-tolerated, effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long-term antiviral therapy, and its integration, though no longer able to support viral replication, destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome, which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products, and thus, it is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets. AU - Hu, J.* AU - Protzer, U. AU - Siddiqui, A.* C1 - 57020 C2 - 47446 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Revisiting hepatitis B virus: Challenges of curative therapies. JO - J. Virol. VL - 93 IS - 20 PB - Amer Soc Microbiology PY - 2019 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Chou, W.M. AU - Mück-Häusl, M. AU - Ko, C. AU - Wettengel, J.M. AU - Protzer, U. AU - Stadler, D. C1 - 53751 C2 - 44997 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - 37-37 TI - Establishment and characterization of a novel recombinant reporter HBV utilizing the Cre-lox recombination system. JO - J. Virol. VL - 25 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) infects the oropharynx but, surprisingly, frequently induces B cell proliferation in the gut of immunosuppressed individuals. We found that EBV infection in vitro induces the expression of the LPAM-1 integrin on tonsillar B cells and increases it on peripheral blood cells. Similarly, LPAM-1 was induced in the tonsils of patients undergoing primary infectious mononucleosis. EBV-induced LPAM-1 bound to the MAdCAM-1 addressin, which allows B cell homing to the gastrointestinal mucosa-associated lymphoid tissue (GALT). Thus, we hypothesized that EBV-induced LPAM-1 could induce relocation of infected B cells from the tonsil to the GALT. In situ hybridization with an EBER-specific probe revealed the frequent presence of EBV-infected cells in the pericolic lymph nodes of healthy individuals. Relocation of infected B cells into the GALT would expand the EBV reservoir, possibly protecting it from T cells primed in the oropharynx, and explain why EBV induces lymphoid tumors in the gut. IMPORTANCE EBV causes tumors in multiple organs, particularly in the oro- and na-sopharyngeal area but also in the digestive system. This virus enters the body in the oropharynx and establishes a chronic infection in this area. The observation that the virus causes tumors in the digestive system implies that the infected cells can move to this organ. We found that EBV infection induces the expression of integrin beta 7 (ITGB7), an integrin that associates with integrin alpha 4 to form the LPAM-1 dimer. LPAM-1 is key for homing of B cells to the gastrointestinal tract, suggesting that induction of this molecule is the mechanism through which EBV-infected cells enter this organ. In favor of this hypothesis, we could also detect EBV-infected cells in the lymph nodes adjacent to the colon and in the appendix. AU - Delecluse, S.* AU - Tsai, M.H.* AU - Shumilov, A.* AU - Bencun, M.* AU - Arrow, S.* AU - Beshirova, A.* AU - Cottignies-Calamarte, A.* AU - Lasitschka, F.* AU - Bulut, O.C.* AU - Münz, C.* AU - Zeier, M.* AU - Behrends, U. AU - Delecluse, H.J.* C1 - 55450 C2 - 46157 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Epstein-Barr virus induces expression of the LPAM-1 integrin in B cells in vitro and in vivo. JO - J. Virol. VL - 93 IS - 5 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Feuerherd, M. AU - Körber, N. AU - Boesecke, C.* AU - Mohrmann, K.* AU - Froeschl, G.* AU - Ringelhan, M. AU - Geisler, F.* AU - Rockstroh, J.* AU - Protzer, U. AU - Bauer, T. C1 - 53750 C2 - 44994 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - 52-53 TI - Altered HBV-specific CD4+T-cell Responses in HBV/HIV-1 Co-versus HBV Mono-infected Patients. JO - J. Virol. VL - 25 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequenceindependent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion as the functions are more diverse. We showed that capsid protein VI regulates the antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, which is beneficial for efficient viral gene expression. Here, we were able to show that core protein V also represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. In conclusion, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency. AU - Freudenberger, N. AU - Meyer, T.* AU - Groitl, P. AU - Dobner, T.* AU - Schreiner, S. C1 - 52399 C2 - 43944 CY - Washington TI - HAdV protein V core protein is targeted by the host SUMOylation machinery to limit essential viral functions. JO - J. Virol. VL - 92 IS - 4 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Since 2014, reclaimed water has been used for agricultural irrigation and it has been discharged on a riverbed of the Maneadero aquifer, Baja California, Mexico. To determine the effects of reclaimed water on groundwater quality, samples of reclaimed water and groundwater were collected spatiotemporal and analyzed using stable isotope (δ18OH2O, δ2HH2O, δ18ONO3and δ15NNO3) and geochemical signatures, jointly with multivariate statistical methods and a 2D resistivity tomography. Reverse ion exchange and mineralization are the main processes influencing the groundwater composition. The Cl/Br ratios identified seawater intrusion and solid waste, wastewater and animal waste as the main sources responsible for these processes, overlapping with the ratios of reclaimed water. Nitrates are pervasive throughout the aquifer and δ18ONO3and δ15NNO3attributed wastewater and animal waste as the major nitrates inputs. Multivariate statistics were able to separate seawater and human-derived processes. The δ18OH2Oand δ2HH2Oshowed the effect of mixing with d-excess of 5–6‰, indicating recharge other than precipitation. A mixing model using Cl−and δ18OH2Oand principal components revealed the mixing proportion of seawater; whilst the over- and under-estimates of reclaimed water contribution are indicative of missing end-members. The Na-Cl-Br-B systematics, however, suggest that reclaimed water result in cation-exchange and adsorption reactions and once the adsorbed sites become saturated with respect of Na+, Br−and B−can be reflected in the groundwater composition. Additionally, resistivities indicate that reclaimed water interacts between the fresh and brackish groundwater. Monitoring the efficiency of the vadose zone to retain contaminants and distinguish them from reclaimed water is essential for evaluating groundwater quality. AU - Gilabert-Alarcon, C. AU - Daessle, L.W.* AU - Salgado-Mendez, S.O.* AU - Perez-Flores, M.A.* AU - Knoeller, K.* AU - Kretzschmar, T.G.* AU - Stumpp, C. C1 - 53756 C2 - 45000 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - 121-139 TI - Effects of reclaimed water discharge in the Maneadero coastal aquifer, Baja California, Mexico. JO - J. Virol. VL - 92 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - González-Hernández, M.* AU - Hoffmann, M.* AU - Brinkmann, C.* AU - Nehls, J. AU - Winkler, M.* AU - Schindler, M. AU - Pöhlmann, S.* C1 - 53708 C2 - 44976 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - A GXXXA motif in the transmembrane domain of the Ebola virus glycoprotein is required for tetherin antagonism. JO - J. Virol. VL - 92 IS - 13 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Ebola virus (EBOV) and Nipah virus (NiV) infection of humans can cause fatal disease and constitutes a public health threat. In contrast, EBOV and NiV infection of fruit bats, the putative (EBOV) or proven (NiV) natural reservoir, is not associated with disease, and it is currently unknown how these animals control the virus. The human interferon (IFN)-stimulated antiviral effector protein tetherin (CD317, BST-2) blocks release of EBOV- and NiV-like particles from cells and is counteracted by the EBOV glycoprotein (GP). In contrast, it is unknown whether fruit bat tetherin restricts virus infection and is susceptible to GP-driven antagonism. Here, we report the sequence of fruit bat tetherin and show that its expression is IFN stimulated and associated with strong antiviral activity. Moreover, we demonstrate that EBOV- GP antagonizes tetherin orthologues of diverse species but fails to efficiently counteract fruit bat tetherin in virus-like particle (VLP) release assays. However, unexpectedly, tetherin was dispensable for robust IFN-mediated inhibition of EBOV spread in fruit bat cells. Thus, the VLP-based model systems mimicking tetherin-mediated inhibition of EBOV release and its counteraction by GP seem not to adequately reflect all aspects of EBOV release from IFN-stimulated fruit bat cells, potentially due to differences in tetherin expression levels that could not be resolved by the present study. In contrast, tetherin expression was essential for IFN-dependent inhibition of NiV infection, demonstrating that IFN-induced fruit bat tetherin exerts antiviral activity and may critically contribute to control of NiV and potentially other highly virulent viruses in infected animals.IMPORTANCE Ebola virus and Nipah virus (EBOV and NiV) can cause fatal disease in humans. In contrast, infected fruit bats do not develop symptoms but can transmit the virus to humans. Why fruit bats but not humans control infection is largely unknown. Tetherin is an antiviral host cell protein and is counteracted by the EBOV glycoprotein in human cells. Here, employing model systems, we show that tetherin of fruit bats displays higher antiviral activity than human tetherin and is largely resistant against counteraction by the Ebola virus glycoprotein. Moreover, we demonstrate that induction of tetherin expression is critical for interferon-mediated inhibition of NiV but, for at present unknown reasons, not EBOV spread in fruit bat cells. Collectively, our findings identify tetherin as an antiviral effector of innate immune responses in fruit bats, which might allow these animals to control infection with NiV and potentially other viruses that cause severe disease in humans. AU - Hoffmann, M.* AU - Nehlmeier, I.* AU - Brinkmann, C.* AU - Krähling, V.* AU - Behner, L.* AU - Moldenhauer, A.S.* AU - Krüger, N.* AU - Nehls, J. AU - Schindler, M. AU - Hoenen, T.* AU - Maisner, A.* AU - Becker, S.* AU - Pöhlmann, S.* C1 - 55021 C2 - 45993 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Tetherin inhibits nipah Vvrus but not ebola virus replication in fruit bat cells. JO - J. Virol. VL - 93 IS - 3 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Karimzadeh, H. AU - Kiraithe, M.M.* AU - Kosinska, A. AU - Glaser, M.* AU - Fiedler, M.* AU - Oberhardt, V.* AU - Alizei, E.S.* AU - Hofmann, M.* AU - Mok, J.Y.* AU - Nguyen, M.* AU - van Esch, W.J.E.* AU - Budeus, B.* AU - Grabowski, J.* AU - Homs, M.* AU - Olivero, A.* AU - Keyvani, H.* AU - Rodríguez-Frías, F.* AU - Tabernero, D.* AU - Buti, M.* AU - Heinold, A.* AU - Alavian, S.M.* AU - Bauer, T. AU - Wiesch, J.S.z.* AU - Raziorrouh, B.* AU - Hoffmann, D.* AU - Smedile, A.* AU - Rizzetto, M.* AU - Wedemeyer, H.* AU - Timm, J.* AU - Antes, I.* AU - Neumann-Haefelin, C.* AU - Protzer, U. AU - Roggendorf, M. C1 - 53707 C2 - 44975 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Amino acid substitutions within HLA-B*27-restricted T cell epitopes prevent recognition by hepatitis delta virus-specific CD8+ T cells. JO - J. Virol. VL - 92 IS - 13 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Menni, C.* AU - Gudelj, I.* AU - Macdonald-Dunlop, E.* AU - Mangino, M.* AU - Zierer, J. AU - Besic, E.* AU - Joshi, P.K.* AU - Trbojević-Akmačić, I.* AU - Chowienczyk, P.J.* AU - Spector, T.D.* AU - Wilson, J.F.* AU - Lauc, G.* AU - Valdes, A.M.* C1 - 53755 C2 - 44999 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - 1555-1564 TI - Glycosylation profile of immunoglobulin G is cross-sectionally associated with cardiovascular disease risk score and subclinical atherosclerosis in two independent cohorts. JO - J. Virol. VL - 122 IS - 11 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Müncheberg, S. AU - Hay, R.T.* AU - Ip, W.H.* AU - Meyer, T.* AU - Weiß, C. AU - Brenke, J.K. AU - Masser, S. AU - Hadian, K. AU - Dobner, T.* AU - Schreiner, S. C1 - 53454 C2 - 44867 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - e00164-18 TI - E1B-55K mediated regulation of RNF4 STUbL promotes HAdV gene expression. JO - J. Virol. VL - 92 IS - 13 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine. AU - Ning, X.* AU - Luckenbaugh, L.* AU - Liu, K.* AU - Bruss, V. AU - Sureau, C.* AU - Hu, J.J.* C1 - 53501 C2 - 44884 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Common and distinct capsid and surface protein requirements for secretion of complete and genome-free hepatitis B virions. JO - J. Virol. VL - 92 IS - 14 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - The cellular protein SPOC1 (survival time-associated PHD [plant homeodomain] finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and the DNA damage response. It binds H3K4me2/3-containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4-orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of human cytomegalovirus (HCMV) replication. We show that the expression of immediate early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection, SPOC1 is downregulated in a glycogen synthase kinase 3 beta (GSK-3 beta)-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed an augmented initiation of viral IE gene expression. This occurs in a multiplicity of infection (MOI)-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection, while later upregulation had no negative impact, suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate early promoter (MIEP), strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and the subsequent recruitment of heterochromatin-building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.IMPORTANCE Accumulating evidence indicates that during millennia of coevolution, host cells have developed a sophisticated compilation of cellular factors to restrict cytomegalovirus infections. Defining this equipment is important to understand cellular barriers against viral infection and to develop strategies to utilize these factors for antiviral approaches. So far, constituents of PML nuclear bodies and interferon gamma-inducible protein 16 (IFI16) were known to mediate intrinsic immunity against HCMV. In this study, we identify the chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that preexisting high SPOC1 protein levels mediate a silencing of HCMV gene expression via a specific association with an important viral cis-regulatory element, the major immediate early promoter. Since SPOC1 expression varies between cell types, this factor may play an important role in tissue-specific defense against HCMV. AU - Reichel, A.* AU - Stilp, A.C.* AU - Scherer, M.* AU - Reuter, N.* AU - Lukassen, S.* AU - Kasmapour, B.* AU - Schreiner, S. AU - Cicin-Sain, L.* AU - Winterpacht, A.* AU - Stamminger, T.* C1 - 53502 C2 - 44885 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Chromatin-remodeling factor SPOC1 acts as a cellular restriction factor against human cytomegalovirus by repressing the major immediate early promoter. JO - J. Virol. VL - 92 IS - 14 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Schreiber, S. AU - Honz, M. AU - Schiemann, M.* AU - Protzer, U. AU - Wisskirchen, K. C1 - 53749 C2 - 44993 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - 47-47 TI - Generating CD4+T cells for the T-cell therapy of HBV infection. JO - J. Virol. VL - 25 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOy lated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4-and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. AU - Stadler, D. AU - Hess, J.* AU - Schneider, J. AU - Lasitschka, F.* AU - Ringelhan, M. AU - Ko, C. AU - Chou, W.M. AU - Wettengel, J.M. AU - Unger, K.* AU - Protzer, U. C1 - 53748 C2 - 44986 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa SP - 17-17 TI - Identification of ISG20 as the nuclease involved in Interferon-induced decline of HBV cccDNA. JO - J. Virol. VL - 25 PB - Amer Soc Microbiology PY - 2018 SN - 0022-538X ER - TY - JOUR AB - During hepatitis B virus (HBV) infections, subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and hydrophobic C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (the cytosolic loop [CL]) and between TM2 and the HCR (the luminal loop [LL]) are located in the cytosol and the endoplasmic reticulum (ER) lumen, respectively. To define the domains of S mediating oligomerization during SVP morphogenesis, S mutants were characterized by expression in transiently transfected cells. Mutation of 12 out of 15 amino acids of TM1 to alanines, as well as the deletion of HCR, still allowed SVP formation, demonstrating that these two domains are not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a fluorescence-activated cell sorter (FACS)-based Förster resonance energy transfer (FRET) assay. This approach demonstrated that the CL, TM2, and the LL independently contributed to S oligomerization, while TM1 and the HCR played minor roles. Apparently, intermolecular homo-oligomerization of the CL, TM2, and the LL drives S protein aggregation. Detailed analyses revealed that the point mutation C65S in the CL, the mutation of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous replacement of all 8 cysteine residues in the LL by serine residues blocked the abilities of these domains to support S protein interactions. Altogether, specific domains and residues in the HBV S protein that are required for oligomerization and SVP generation were defined. AU - Suffner, S. AU - Gerstenberg, N. AU - Patra, M. AU - Ruibal, P. AU - Orabi, A. AU - Schindler, M. AU - Bruss, V. C1 - 53262 C2 - 44519 TI - Domains of the hepatitis B virus small surface protein S mediating oligomerization. JO - J. Virol. VL - 92 IS - 11 PY - 2018 SN - 0022-538X ER - TY - JOUR AB - More than 90% of humanity is infected with Epstein-Barr virus (EBV) lifelong. While infection is usually controlled by the immune system, the human host fails to completely eliminate the pathogen. Several herpesviral proteins are known that act as immunoevasins preventing or reducing recognition of EBV-infected cells. Only recently, microRNAs of EBV were identified to reduce immune recognition further. This Gem summarizes what we know about immunomodulatory miRNAs of herpesviruses. AU - Albanese, M. AU - Tagawa, T. AU - Buschle, A. AU - Hammerschmidt, W. C1 - 51278 C2 - 43169 CY - Washington TI - MicroRNAs of Epstein-Barr virus control innate and adaptive anti-viral immunity. JO - J. Virol. VL - 91 IS - 16 PB - Amer Soc Microbiology PY - 2017 SN - 0022-538X ER - TY - JOUR AB - Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation, because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since MKRN1 protein accumulation is also reduced in measles virus-and vesicular stomatitis virus-infected cells, our results signify the general strategy of viruses to target MKRN1. AU - Inturi, R.* AU - Mun, K.* AU - Singethan, K. AU - Schreiner, S. AU - Punga, T.* C1 - 52357 C2 - 43928 CY - Washington TI - Human adenovirus infection causes cellular E3 ubiquitin ligase MKRN1 degradation involving the viral core protein pVII. JO - J. Virol. VL - 92 IS - 3 PB - Amer Soc Microbiology PY - 2017 SN - 0022-538X ER - TY - JOUR AB - Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169(+) cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169(+) cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b(+) Ly6C(+) Ly6G(+) cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169(+) cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169(+) cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169(+) cells and innate and adaptive immune activation during VSV infection. IMPORTANCE Over the last decade, strategically placed CD169(+) metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169(+) macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169(+) macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169(+) macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance. AU - Shinde, P.V.* AU - Xu, H.C.* AU - Maney, S.K.* AU - Kloetgen, A.* AU - Namineni, S.* AU - Zhuang, Y.* AU - Honke, N.* AU - Shaabani, N.* AU - Bellora, N.* AU - Doerrenberg, M.* AU - Trilling, M.* AU - Pozdeev, V.I.* AU - van Rooijen, N.* AU - Scheu, S.* AU - Pfeffer, K.* AU - Crocker, P.R.* AU - Tanaka, M.* AU - Duggimpudi, S.* AU - Knolle, P. AU - Heikenwalder, M.* AU - Ruland, J.* AU - Mak, T.W.* AU - Brenner, D.* AU - Pandyra, A.A.* AU - Hoell, J.I.* AU - Borkhardt, A.* AU - Häussinger, D.* AU - Lang, K.S.* AU - Lang, P.A.* C1 - 52356 C2 - 43929 CY - Washington TI - Tumor necrosis factor-mediated survival of CD169+ cells promotes immune activation during vesicular stomatitis virus infection. JO - J. Virol. VL - 92 IS - 3 PB - Amer Soc Microbiology PY - 2017 SN - 0022-538X ER - TY - JOUR AB - We analyzed HCV morphogenesis using viral genomes encoding for a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization and replication kinetics were comparable to untagged HCV and E1-mCherry tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and non-structural NS5A followed a tempo-spatial pattern with succinct decrease in replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating absence of viral glycoprotein processing by the Golgi. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggests that assembled HCV particles are released via a non-canonical secretory route involving the endosomal compartment. IMPORTANCE STATEMENT: The goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which result in the production of fluorescently labeled viral particles. We used live cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap with Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point towards the possible existence of a not yet described cellular secretion route. AU - Bayer, K. AU - Banning, C.* AU - Bruss, V. AU - Wiltzer-Bach, L.* AU - Schindler, M. C1 - 49493 C2 - 40703 CY - Washington SP - 10558-10573 TI - Hepatitis C virus is released via a non-canonical secretory route. JO - J. Virol. VL - 90 IS - 23 PB - Amer Soc Microbiology PY - 2016 SN - 0022-538X ER - TY - JOUR AB - The glycoprotein of Ebola virus (EBOV-GP), a member of the Filoviridae family, facilitates viral entry into target cells. In addition, EBOV-GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV-release from infected cells. However, it is unclear how EBOV-GP antagonizes tetherin and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV-GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV-GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV-GP is a prerequisite to tetherin counteraction. In contrast, blockade of Niemann-Pick disease, type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data in conjunction with previous reports indicate that tetherin antagonism is conserved between the GPs of all known filoviruses and demonstrate that the GP1 subunits of EBOV-GP plays a central role in tetherin antagonism. IMPORTANCE: Filoviruses are re-emerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in Western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in Northern Spain, is inhibited by innate antiviral effectors in human cells might help to define whether the virus constitutes a threat to humans. The present study shows that LLOV like EBOV counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV-GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction. AU - Brinkmann, C.* AU - Nehlmeier, I.* AU - Walendy-Gnirß, K.* AU - Nehls, J. AU - González Hernández, M.* AU - Hoffmann, M.* AU - Qiu, X.* AU - Takada, A.* AU - Schindler, M. AU - Pöhlmann, S.* C1 - 49693 C2 - 40867 CY - Washington SP - 11075-11086 TI - Tetherin antagonism by the Ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by GP1-specific antibodies. JO - J. Virol. VL - 90 IS - 24 PB - Amer Soc Microbiology PY - 2016 SN - 0022-538X ER - TY - JOUR AB - Interleukin 2 (IL2) signaling through the IL2 receptor alpha chain+ (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25+FoxP3+CD4+ T cells. CD25+FoxP3+CD4+ T cells may therefore constitute a suitable subset for HIV infection and plasma virion production.CD25+FoxP3+CD4+ T cell frequencies, absolute numbers and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV+ and HIV- study volunteers. Different memory CD4+ T cell subsets were then sorted for quantification of cell-associated HIV-DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences.In HIV+ subjects, 51% (median) of CD25+FoxP3+CD4+ T cells expressed the HIV co-receptor CCR5. Very high frequencies of Ki67+ cells were detected in CD25+FoxP3+ (median, 27.6%) in comparison to memory CD25-FoxP3- memory CD4+ T cells (median, 4.1%, p<0.0001). HIV-DNA content was 15-fold higher in CD25+FoxP3+ compared to CD25-FoxP3- memory CD4+ T cells (p=0.003). EnvV1V3 sequences derived from CD25+FoxP3+ memory CD4+ T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma-sequence pairs were rare and their proportion further decreased with the estimated HIV infection duration.These data suggest that specific cellular characteristics of CD25+FoxP3+ memory CD4+ T cell might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. Contribution of this cell population to plasma virion production remains unclear. IMPORTANCE: Despite recent advances in the understanding of AIDS virus pathogenesis, it is incompletely understood, which cell subsets support HIV infection and replication in vivo In vitro, the IL2 signaling pathway and IL2 dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25+FoxP3+ memory CD4 T cells - often referred to as regulatory CD4 T cells - depend on IL2 signaling for homeostatic proliferation in vivo Our results show that CD25+FoxP3+ memory CD4+ T cells often express the HIV co-receptor CCR5, are significantly more proliferative and contain more HIV-DNA compared to CD25-FoxP3- memory CD4 T cell subsets. The specific cellular characteristics of CD25+FoxP3+ memory CD4+ T cell probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However contribution of this cell subset to plasma viremia remains unclear. AU - Chachage, M.* AU - Pollakis, G.* AU - Kuffour, E.O.* AU - Haase, K.* AU - Bauer, A.* AU - Nadai, Y.* AU - Podola, L.* AU - Clowes, P.* AU - Schiemann, M. AU - Henkel, L. AU - Hoffmann, D. AU - Joseph, S.S.* AU - Bhuju, S.* AU - Maboko, L.* AU - Sarfo, F.S.* AU - Eberhardt, K.* AU - Hoelscher, M.* AU - Feldt, T.* AU - Saathoff, E.* AU - Geldmacher, C.* C1 - 49035 C2 - 41563 CY - Washington SP - 8954-8967 TI - CD25+ FoxP3+ memory CD4 T cells are frequent targets of HIV infection in vivo. JO - J. Virol. VL - 90 IS - 20 PB - Amer Soc Microbiology PY - 2016 SN - 0022-538X ER - TY - JOUR AB - Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. The MWPyV small T antigen (ST) binds protein phosphatase 2A (PP2A), and the large T antigen (LT) binds pRb, p107, p130, and p53. However, the MWPyV LT was less stable than the simian virus 40 (SV40) LT and was unable to promote the growth of normal cells. This report confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential. AU - Berrios, C.* AU - Jung, J.U.* AU - Primi, B.* AU - Wang, M.* AU - Pedamallu, C.* AU - Duke, F.* AU - Marcelus, C.* AU - Cheng, J.* AU - Garcea, R.L.* AU - Meyerson, M.* AU - DeCaprio, J.A.* AU - TEDDY Study Group (Ziegler, A.-G. AU - Beyerlein, A. AU - Henneberger, L. AU - Hummel, M. AU - Hummel, S. AU - Knopff, A. AU - Krause, S. AU - Peplow, C. AU - Pflüger, M. AU - Roth, R. AU - Schenkel, J. AU - Stock, J. AU - Strauss, E. AU - Warncke, K. AU - Winkler, C.) C1 - 44203 C2 - 39400 CY - Washington SP - 857-862 TI - Malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors. JO - J. Virol. VL - 89 IS - 1 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - Once transported to the replication sites, HAdVs need to assure decondensation and transcriptional activation of their viral genomes to synthesize viral proteins and initiate steps to reprogram the host cell for viral replication. These early stages during adenoviral infection are poorly characterized, but represent a decisive moment in establishing a productive infection. Here, we identify a novel host viral restriction factor, KAP1. This heterochromatin associated transcription factor regulates the dynamic organization of host chromatin structure via its ability to influence epigenetic marks and chromatin compaction. In response to DNA damage, KAP1 is phosphorylated and functionally inactive, resulting in chromatin relaxation. We discovered that KAP1 posttranslational modification is dramatically altered during HAdV infection to limit the antiviral capacity of this host restriction factor, which represents an essential step required for efficient viral replication. Conversely, we also observed an HAdV-mediated decrease of KAP1 SUMO moieties during infection, known to promote chromatin-decondensation events. Based on our findings, we provide evidence that HAdV induces KAP1 deSUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism. IMPORTANCE: Here we describe a novel cellular restriction factor for Human Adenovirus (HAdV) that sheds light on very early modulation processes in viral infection. We reported that chromatin formation and cellular SWI/SNF chromatin remodeling play a key role in HAdV transcriptional regulation (1-4). We observed that the cellular chromatin-associated factor, and epigenetic reader SPOC1 represses HAdV infection and gene expression. Here, we illustrate the role of the SPOC1 interacting factor KAP1 during productive HAdV growth. KAP1 binds to the viral E1B-55K protein, promoting its SUMO modification, therefore illustrating a crucial step for efficient viral replication. Simultaneously, KAP1 posttranslational modification is dramatically altered during infection. We observed an HAdV-mediated decrease in KAP1 SUMOylation, known to promote chromatin-decondensation events. These findings indicate that HAdV induces loss of KAP1 SUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism. AU - Bürck, C.* AU - Mund, A.* AU - Berscheminski, J.* AU - Kieweg, L.* AU - Müncheberg, S. AU - Dobner, T.* AU - Schreiner, S. C1 - 47218 C2 - 39185 SP - 930-946 TI - KAP1 is a host restriction factor that promotes HAdV E1B-55K SUMO modification. JO - J. Virol. VL - 90 IS - 2 PY - 2015 SN - 0022-538X ER - TY - JOUR AB - Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates, envelopment by viral envelope proteins leading to secretion extracellularly as virions or disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or didn't significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCE: Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete virion particles, or alternatively, can deliver their DNA to host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of these same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes. AU - Cui, X.* AU - Luckenbaugh, L.* AU - Bruss, V. AU - Hu, J.J.* C1 - 46413 C2 - 37550 CY - Washington SP - 10064-10072 TI - Alteration of mature nucleocapsid and enhancement of covalently closed circular DNA formation by Hepatitis B Virus core mutants defective in complete virion formation. JO - J. Virol. VL - 89 IS - 19 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - The expression of the antiviral host cell factor tetherin is induced by interferon and can inhibit the release of enveloped viruses from infected cells. The Vpu protein of HIV-1 antagonizes the antiviral activity of tetherin and tetherin antagonists with Vpu-like activity have been identified in other viruses. In contrast, it is incompletely understood whether tetherin inhibits influenza A virus (FLUAV) release and whether FLUAV encodes tetherin antagonists. Here, we show that release of several laboratory-adapted and a seasonal FLUAV strain is inhibited by tetherin while pandemic FLUAV A/Hamburg/4/2009 is resistant. Studies with a virus-like particle system and analysis of reassortant viruses provided evidence that the viral hemagglutinin (HA) is an important determinant of tetherin antagonism but requires the presence of its cognate neuraminidase (NA) to inhibit tetherin. Finally, tetherin antagonism by FLUAV was dependent on the virion context, since retrovirus release from tetherin-positive cells was not rescued, and correlated with a HA, NA-dependent reduction in tetherin expression. In sum, our study identifies HA and NA proteins of certain pandemic FLUAV as tetherin antagonists, which has important implications for understanding FLUAV pathogenesis. IMPORTANCE: Influenza A virus (FLUAV) infection is responsible for substantial global morbidity and mortality and understanding how the virus evades immune defenses of the host may uncover novel targets for antiviral intervention. Tetherin is an antiviral effector molecule of the innate immune system which can contribute to control of viral invasion. However, it has been unclear whether FLUAV are inhibited by tetherin and whether these viruses encode tetherin antagonizing proteins. Our observation that several pandemic FLUAV can counteract tetherin via their HA and NA proteins identifies these proteins as novel tetherin antagonists and indicates that HA/NA-dependent inactivation of innate defenses may contribute to the efficient spread of pandemic FLUAV. AU - Gnirß, K.* AU - Zmora, P.* AU - Blazejewska, P.* AU - Winkler, M.* AU - Lins, A.* AU - Nehlmeier, I.* AU - Gärtner, S.* AU - Moldenhauer, A.S.* AU - Hofmann-Winkler, H.* AU - Wolff, T.* AU - Schindler, M. AU - Pöhlmann, S.* C1 - 45473 C2 - 37355 SP - 9178-9188 TI - Tetherin sensitivity of influenza A viruses is strain specific: Role of hemagglutinin and neuraminidase. JO - J. Virol. VL - 89 IS - 18 PY - 2015 SN - 0022-538X ER - TY - JOUR AB - HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, β-COP and ACOT8 for CD4 downmodulation in HIV-1 infected shRNA-expressing CD4+ T cells and characterized direct interaction with Nef by FRET. Binding of lentiviral Nefs to CD4 and AP-2 was conserved and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation. AU - Gondim, M.V. AU - Wiltzer-Bach, L.* AU - Maurer, B.* AU - Banning, C.* AU - Arganaraz, E.* AU - Schindler, M. C1 - 47262 C2 - 39305 SP - 12518-12524 TI - AP-2 is the crucial clathrin adaptor protein for CD4 downmodulation by HIV-1 Nef in primary infected CD4+ T cells. JO - J. Virol. VL - 89 IS - 24 PY - 2015 SN - 0022-538X ER - TY - JOUR AB - CD8(+) T cells are main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems such as low infection rates restricted mechanistic studies of HBV-specific CD8(+) T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2(hNTCP) cells that endogenously processed and presented viral antigens to HBV-specific CD8(+) T cells. This induced cytolytic and non-cytolytic CD8(+) T-cell effector functions and reduction of viral loads. AU - Hoh, A.* AU - Heeg, M.* AU - Ni, Y.* AU - Schuch, A.* AU - Binder, B.* AU - Hennecke, N.* AU - Blum, H.E.* AU - Nassal, M.* AU - Protzer, U. AU - Hofmann, M.* AU - Urban, S.* AU - Thimme, R.* C1 - 44865 C2 - 37182 CY - Washington SP - 7433-7438 TI - HBV-infected HepG2hNTCP cells serve as a novel immunological tool to analyze the antiviral efficacy of CD8+ T cells in vitro. JO - J. Virol. VL - 89 IS - 14 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/Tetherin elimination from the surface of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surface of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment. IMPORTANCE: The HIV-1 accessory protein Vpu has previously been shown to downregulate various host-cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surface of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness. AU - Lambelé, M.* AU - Koppensteiner, H. AU - Symeonides, M.* AU - Roy, N.H.* AU - Chan, J.* AU - Schindler, M. AU - Thali, M.* C1 - 43074 C2 - 35994 CY - Washington SP - 3247-3255 TI - Vpu is the main determinant for tetraspanin downregulation in HIV-1 infected cells. JO - J. Virol. VL - 89 IS - 6 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain, MD) in the large surface protein L and a narrow region (matrix binding domain, MBD) including isoleucine 126 on the capsid surface have been mapped where point mutations like core-I126A specifically blocked nucleocapsid envelopment. Possibly, both domains interact with each other during virion morphogenesis. By the SELEX method we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50 % of the clones. The main aptamer AO-01 (13 clones, 18 %) showed the lowest dissociation constant (Kd) of 180 +/- 82 nM for capsid binding among the four molecules. Its Kd value for I126A capsids was 1306 +/- 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47 % inhibition of virion production 3 days post transfection but showed no inhibition by cotransfection of an aptamer with random sequence. The half-life of AO-01 in cells was 2 hours which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction by a low molecular weight substance might represent a new approach for an antiviral therapy. IMPORTANCE: Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55 base long single stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The result substantiates the current model for HBV morphogenesis and shows that the capsid envelope interaction is a potential antiviral target. AU - Orabi, A. AU - Bieringer, M. AU - Geerlof, A. AU - Bruss, V. C1 - 45677 C2 - 37425 SP - 9281-9287 TI - An aptamer against the matrix binding domain on the hepatitis B virus capsid impairs virion formation. JO - J. Virol. VL - 89 IS - 18 PY - 2015 SN - 0022-538X ER - TY - JOUR AB - The human herpes viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpes virus (KSHV) are associated with Hodgkin's lymphoma (HL) and Primary effusion lymphomas (PEL), respectively, which are B cell malignancies that originate from germinal center B cells. PEL cells but also a quarter of EBV-positive HL tumor cells do not express the genuine B cell receptor (BCR), a situation incompatible with survival of normal B cells. EBV encodes LMP2A, one of EBV's viral latent membrane proteins, which likely replaces the BCR's survival signaling in HL. Whether KSHV encodes a viral BCR mimic that contributes to oncogenesis is not known because an experimental model of KSHV-mediated B cell transformation is lacking. We addressed this uncertainty with mutant EBVs encoding the KSHV genes K1 or K15 in lieu of LMP2A and infected primary BCR-negative (BCR(-)) human B cells with them. We confirmed that the survival of BCR(-) B cells and their proliferation depended on an active LMP2A signal. Like LMP2A, expression of K1 and K15 led to the survival of BCR(-) B cells prone to apoptosis, supported their proliferation and regulated a similar set of cellular target genes. K1 and K15 encoded proteins appear to have non-complementing, redundant functions in this model but our findings suggest that both KSHV proteins can replace LMP2A's key activities contributing to the survival, activation and proliferation of BCR(-) PEL cells in vivo. IMPORTANCE: Several herpes viruses encode oncogenes that are receptor-like proteins. Often, they are constitutively active providing important functions to the latently infected cells. LMP2A of Epstein-Barr virus (EBV) is such a receptor that mimics an activated B cell receptor, BCR. K1 and K15, related receptors of Kaposi sarcoma-associated herpes virus (KSHV) expressed in virus-associated tumors, have less obvious functions. We found in infection experiments that both viral receptors of KSHV can replace LMP2A and deliver functions similar to the endogenous BCR. K1, K15, and LMP2A also control the expression of a related set of cellular genes in primary human B cells, the target cells of EBV and KSHV. The observed phenotypes as well as the known characteristics of these genes argue for their contributions to cellular survival, B cell activation, and proliferation. Our findings provide one possible explanation for the tumorigenicity of KSHV, which poses a severe problem in immunocompromised patients. AU - Steinbrück, L. AU - Gustems, M. AU - Medele, S. AU - Schulz, T.F.* AU - Lutter, D. AU - Hammerschmidt, W. C1 - 44800 C2 - 37041 CY - Washington SP - 7248-7261 TI - K1 and K15 of Kaposi sarcoma-associated herpes virus are partial functional homologues of latent membrane protein 2A of Epstein-Barr virus. JO - J. Virol. VL - 89 IS - 14 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - CD4+ T-lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on MHCII molecules on antigen presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4+ T-cells. Certain epitopes, however, can be directly processed from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways being possibly involved in the immune response upon vaccination with MVA (modified vaccinia virus Ankara), a promising live viral vaccine vector. We established CD4+ T-cell lines specific for MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4+ T-cells. By using knockout mice and chemical inhibitory compounds we further elucidate the molecular basis showing that among the various subcellular pathways investigated proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role neither TAP nor LAMP-2 were found to be involved in the peptide transport. Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis to improve MVA-based vaccination strategies aiming for enhanced CD4+ T-cell activation by targeting antigens into the responsible pathways. IMPORTANCE STATEMENT: This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4+ T-cells. We identified autophagosome formation, proteasomal activity and lysosomal integrity to be crucial for endogenous CD4+ T-cell activation. Since poxvirus vectors such as MVA are already used in clinical trials as recombinant vaccines, the data provide important information for the future design of optimized poxviral vaccines for the study of advanced immunotherapy options. AU - Thiele, F. AU - Tao, S.* AU - Zhang, Y. AU - Muschaweckh, A. AU - Zollmann, T.* AU - Protzer, U. AU - Abele, R.* AU - Drexler, I.* C1 - 43039 C2 - 35959 CY - Washington SP - 2698-2709 TI - MVA-infected dendritic cells present CD4+ T-cell epitopes by endogenous MHC class II presentation pathways. JO - J. Virol. VL - 98 IS - 5 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - Leukocytes recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly down-regulated on primary CD4(+) T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell-surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments thereby impeding CD62L transport to the plasma membrane. Besides, Nef decreased total CD62L protein levels. Importantly, infection with wild type but not Nef- and Vpu-deficient HIV-1 inhibited the capacity of primary CD4(+) T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and co-stimulatory signals triggered in primary CD4(+) T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses. IMPORTANCE: L-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocytes homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses against pathogens. Here we report that CD62L is down-modulated on the surface of HIV-1-infected T cells through the activity of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules to reach the plasma membrane. We provide evidence that CD62L down-regulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell-surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences such as systemic viral spread and evasion of the host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis. AU - Vassena, L.* AU - Giuliani, E.* AU - Koppensteiner, H. AU - Bolduan, S. AU - Schindler, M. AU - Doria, M.* C1 - 44071 C2 - 36794 CY - Washington SP - 5687-5700 TI - HIV-1 NEF and VPU interfere with L-selectin (CD62L) cell surface expression to inhibit adhesion and signaling in infected CD4+ t lymphocytes. JO - J. Virol. VL - 89 IS - 10 PB - Amer Soc Microbiology PY - 2015 SN - 0022-538X ER - TY - JOUR AB - The human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, establishes lifelong latency upon infection. Murine gammaherpesvirus 68 (MHV68) is a well-established model for KSHV. Toll-like receptors (TLRs) play a crucial role for the innate immune response to pathogens. Although KSHV and MHV68 are detected by TLRs, studies suggest they modulate TLR4 and TLR9 signaling, respectively. In this study we show that in bone marrow-derived macrophages (BMDMs), MHV68 did not induce a detectable proinflammatory cytokine response. Furthermore, MHV68 abrogated the response to TLR2, 4, 7, and 9 agonists in BMDMs. Similarly to observations with MHV68, infection with KSHV efficiently inhibited TLR2 signaling in THP-1 monocytes. Using a KSHV ORF library, we found that K4.2, ORF21, ORF31, and RTA/ORF50 inhibited TLR2-dependent nuclear factor kappa B (NF-κB) activation in HEK293 TLR2-YFP and Flag-TLR2 transfected HEK 293T cells. Of the identified ORFs, the replication and transcription activator protein, RTA/ORF50, strongly downregulated TLR2 and TLR4 signaling by reducing TLR2 and TLR4 protein expression. Confocal microscopy revealed that TLR2 and TLR4 were no longer localized to the plasma membrane in cells expressing RTA/ORF50. In this study, we have shown that the gammaherpesviruses MHV68 and KSHV efficiently downmodulate TLR signaling in macrophages and have identified a novel function of RTA/ORF50 in modulation of the innate immune response. Importance Statement The Toll-like receptors (TLR) are an important class of pattern recognition receptors of the innate immune system. They induce a potent proinflammatory cytokine response upon detection of a variety of pathogens. In this study, we found that the gammaherpesviruses murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently inhibit the TLR-mediated innate immune response. We further identified the KSHV-encoded replication and transcription activator protein (RTA) as a novel modulator of TLR signaling. Our data suggest that the gammaherpesviruses MHV68 and KSHV prevent activation of the innate immune response by targeting TLR signaling. AU - Bussey, K.* AU - Reimer, E.* AU - Todt, H.* AU - Denker, B.* AU - Gallo, A.* AU - Konrad, A.* AU - Ottinger, M.* AU - Adler, H. AU - Stürzl, M.* AU - Brune, W.* AU - Brinkmann, M.M.* C1 - 31570 C2 - 34554 CY - Washington SP - 9245-9259 TI - The gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and murine gammaherpesvirus 68 modulate the toll-like receptor-induced proinflammatory cytokine response. JO - J. Virol. VL - 88 IS - 16 PB - Amer Soc Microbiology PY - 2014 SN - 0022-538X ER - TY - JOUR AB - The human cytomegalovirus (CMV) UL11 open reading frame (ORF) encodes a putative type I transmembrane glycoprotein, which displays remarkable amino acid sequence variability among different CMV isolates, suggesting that it represents an important virulence factor. In a previous study we have shown that UL11 can interact with the cellular receptor tyrosine phosphatase CD45, which has a central role for signal transduction in T cells, and treatment of T cells with high amounts of a soluble UL11 protein inhibited their proliferation. In order to analyze UL11 expression in CMV-infected cells, we constructed CMV recombinants that either encode tagged UL11 versions or carry a stop mutation in the UL11 ORF. Moreover, we examined whether UL11 affects the function of virus-specific cytotoxic T lymphocytes (CTL). We found that the UL11 ORF gives rise to several proteins due to both posttranslational modification and alternative translation initiation sites. Biotin labelling of surface proteins on infected cells indicated that only highly glycosylated UL11 forms are present at the plasma membrane, whereas low glycosylated UL11 forms were found in the endoplasmic reticulum. We did not find evidence of UL11 cleavage and secretion of a soluble UL11 version. Co-cultivation of CTLs recognizing different CMV epitopes with fibroblasts infected with a UL11 deletion mutant or the parental strain revealed that under the conditions applied UL11 did not influence the activation of CMV-specific CD8 T cells. For further studies we propose to investigate the interaction of UL11 with CD45 and the functional consequences in other immune cells expressing CD45. IMPORTANCE: Human cytomegalovirus belongs to those viruses that extensively interfere with the host immune response. Yet, the precise function of many putative immunomodulatory CMV proteins remains elusive. Previously, we have shown that the CMV UL11 protein interacts with the leukocyte common antigen CD45, a cellular receptor tyrosine phosphatase with a central role for signal transduction in T cells. Here, we examined the proteins expressed by the UL11 gene in CMV infected cells and found that at least one form of UL11 is present at the cell surface, enabling it to interact with CD45 on immune cells. Surprisingly, CMV-expressed UL11 did not affect the activity of virus-specific CD8 T cells. This finding warrants to investigate the impact of UL11 on CD45 functions in other leukocyte subpopulations. AU - Gabaev, I.* AU - Elbasani, E.* AU - Ameres, S. AU - Steinbrück, L.* AU - Stanton, R.* AU - Döring, M.* AU - Lenac Rovis, T.* AU - Kalinke, U.* AU - Jonjic, S.* AU - Moosmann, A. AU - Messerle, M.* C1 - 32430 C2 - 35088 SP - 14326-14339 TI - Expression of the human cytomegalovirus UL11 glycoprotein in viral infection and evaluation of its effect on virus-epecific CD8 T cells. JO - J. Virol. VL - 88 IS - 24 PY - 2014 SN - 0022-538X ER - TY - JOUR AB - Modified Vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses including cell migration. Previously we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells but not neutrophils to the lung. Here we identified neutrophil-attracting chemokines produced by MVA infected primary murine lung fibroblasts and murine bone marrow derived macrophages. We demonstrate that MVA but not vaccinia virus (VACV) strain WR induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1(-/-) mice with MVA as well as application of the CCR1 antagonist J-113863 revealed a role for CCR1 in leukocyte recruitment including neutrophils into the lung. IMPORTANCE: Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA induced leukocyte migration, particularly regarding neutrophils, that could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors. AU - Price, P.J.* AU - Luckow, B.* AU - Torres-Domínguez, L.E.* AU - Brandmüller, C.* AU - Zorn, J. AU - Kirschning, C.J.* AU - Sutter, G.* AU - Lehmann, M.H.* C1 - 31749 C2 - 34734 CY - Washington SP - 10840-10850 TI - Chemokine (C-C motif) receptor 1 (CCR1) is required for efficient recruitment of neutrophils during respiratory infection with Modified Vaccinia virus Ankara. JO - J. Virol. VL - 88 IS - 18 PB - Amer Soc Microbiology PY - 2014 SN - 0022-538X ER - TY - JOUR AB - Vector-borne flaviviruses such as tick-borne encephalitis virus (TBEV), West Nile virus and dengue virus cause millions of infections in humans. TBEV causes a broad range of pathological symptoms ranging from meningitis to severe encephalitis or even hemorrhagic fever with high mortality. Despite the availability of an effective vaccine, incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuro-inflammation in LGTV-infected mice characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that a coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections, however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system is poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection: first in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). Secondly, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis. AU - Weber, E.* AU - Finsterbusch, K.* AU - Lindquist, R.* AU - Nair, S.* AU - Lienenklaus, S.* AU - Gekara, N.O.* AU - Janik, D. AU - Weiss, S.* AU - Kalinke, U.* AU - Overby, A.K.* AU - Kröger, A.* C1 - 31929 C2 - 34905 CY - Washington SP - 12202-12212 TI - Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local anti-viral response. JO - J. Virol. VL - 88 IS - 21 PB - Amer Soc Microbiology PY - 2014 SN - 0022-538X ER - TY - JOUR AB - The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription. AU - Benhenda, S.* AU - Ducroux, A.* AU - Rivière, L.* AU - Sobhian, B.* AU - Ward, M.D.* AU - Dion, S.* AU - Hantz, O.* AU - Protzer, U. AU - Michel, M.L.* AU - Benkirane, M.* AU - Semmes, O.J.* AU - Buendia, M.A.* AU - Neuveut, C.* C1 - 23819 C2 - 31289 SP - 4360-4371 TI - Methyltransferase PRMT1 is a binding partner of HBx and a negative regulator of Hepatitis B Virus transcription. JO - J. Virol. VL - 87 IS - 8 PB - Amer. Soc. Microbiology PY - 2013 SN - 0022-538X ER - TY - JOUR AU - Lankisch, P.* AU - Adler, H. AU - Borkhardt, A.* C1 - 22986 C2 - 30971 SP - 3616-3617 TI - Testing for herpesvirus infection is essential in children with chromosomal-instability syndromes. JO - J. Virol. VL - 87 IS - 6 PB - American Society of Microbiology PY - 2013 SN - 0022-538X ER - TY - JOUR AB - The hepatitis B virus (HBV) surface proteins not only are incorporated into the virion envelope but in addition form subviral particles (SVP) consisting solely of surface proteins and lipids. Heterologous expression of the small HBV envelope protein S produces secreted spherical SVP 20 nm in diameter, with approximately 100 S molecules per particle. The pathway leading from the initial S translation product as a multispanning transmembrane protein to the final SVP is largely unknown. To investigate the role of the four transmembrane domains (TM) of S in this process, we introduced mutations in these regions and characterized their effects on SVP formation in transfected Huh7 cells. We found that the insertion of one amino acid in the center of the alpha-helix of TM1 or the exchange of TM1 with a heterologous TM blocked SVP release and SVP formation by coexpressed wild-type S chains in a transdominant negative fashion. Surprisingly, this effect was partially neutralized when the mutations were expressed in the background of the HBV surface protein M, suggesting that mutations in TM1 could partially be complemented by the pre-S2 domain. The exchange of TM2 with heterologous TMs that form alpha-helices of the same lengths was also incompatible with SVP formation. However, these mutants no longer blocked SVP formation by coexpressed wild-type S. We conclude that TM2 is essential for the stable assembly of S chains by establishing intramembrane interactions. AU - Siegler, V.D. AU - Bruß, V. C1 - 22654 C2 - 30939 SP - 1491-1496 TI - Role of transmembrane domains of hepatitis B virus small surface proteins in subviral-particle biogenesis. JO - J. Virol. VL - 87 IS - 3 PB - American Society of Microbiology PY - 2013 SN - 0022-538X ER - TY - JOUR AB - Human gammaherpesviruses cause morbidity and mortality associated with infection and transformation of lymphoid and endothelial cells. Knowledge of cell types involved in virus dissemination from primary virus entry to virus latency is fundamental for the understanding of gammaherpesvirus pathogenesis. However, the inability to directly trace cell types with respect to virus dissemination pathways has prevented definitive conclusions regarding the relative contribution of individual cell types. Here, we describe that the route of infection affects gammaherpesvirus dissemination pathways. We constructed a recombinant murine gammaherpesvirus 68 (MHV-68) variant harboring a cassette which switches fluorescent markers in a Cre-dependent manner. Since the recombinant virus which was constructed on the wild-type background was attenuated, in this study we used an M1-deleted version, which infected mice with normal kinetics. Infection of Cre-transgenic mice with this convertible virus was used to estimate the quantitative contribution of defined cell types to virus productivity and dissemination during the acute phase of MHV-68 infection. In systemic infection, we found splenic vascular endothelial cells (EC) among the first and main cells to produce virus. After local infection, the contribution of EC to splenic virus production did not represent such early kinetics. However, at later time points, B cell-derived viruses dominated splenic productivity independently of systemic or local infection. Systemic versus local infection also governed the cell types involved in loading peritoneal exudate cells, leading to latency in F4/80- and CD11b-positive target cells. Systemic infection supported EC-driven dissemination, whereas local infection supported B cell-driven dissemination. AU - Vidy, A.* AU - Sacher, T.* AU - Adler, H. AU - Jordan, S.* AU - Koszinowski, U.H.* AU - Ruzsics, Z.* C1 - 23699 C2 - 31245 SP - 4596-4608 TI - Systemic and local infection routes govern different cellular dissemination pathways during gammaherpesvirus infection in vivo. JO - J. Virol. VL - 87 IS - 8 PB - Amer. Soc. of Microbiology PY - 2013 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a human herpesvirus which has been studied intensively for its role in certain human tumors. It also serves as a model of herpesviral latency because it establishes an immediate, latent infection in human B cells. When EBV infe AU - Kalla, M. AU - Göbel, C. AU - Hammerschmidt, W. C1 - 8037 C2 - 29958 SP - 447-458 TI - The lytic phase of Epstein-Barr virus requires a viral genome with 5-methylcytosine residues in CpG sites. JO - J. Virol. VL - 86 IS - 1 PB - Amer. Soc. Microbiology PY - 2012 SN - 0022-538X ER - TY - JOUR AB - In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir. AU - Koppensteiner, H.* AU - Banning, C.* AU - Schneider, C.* AU - Hohenberg, H.* AU - Schindler, M. C1 - 7280 C2 - 29640 SP - 2826-2836 TI - Macrophage internal HIV-1 is protected from neutralizing antibodies. JO - J. Virol. VL - 86 IS - 5 PB - Amer. Soc. Microbiology PY - 2012 SN - 0022-538X ER - TY - JOUR AB - The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5(-/-) mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle. AU - Schneider, M. AU - Ackermann, K.* AU - Stuart, M.* AU - Wex, C.* AU - Protzer, U. AU - Schatzl, H.M.* AU - Gilch, S.* C1 - 10637 C2 - 30952 SP - 10112-10122 TI - Severe acute respiratory syndrome coronavirus replication is severely impaired by MG132 due to proteasome-independent inhibition of M-calpain. JO - J. Virol. VL - 86 IS - 18 PB - Amer. Soc. of Microbiology PY - 2012 SN - 0022-538X ER - TY - JOUR AB - Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus strain, currently under evaluation as a vaccine vector in various clinical settings. It has been reported that human dendritic cells (DCs) mature after infection with MVA, but reports on the functionality of DCs have so far been controversial. In this work, we studied the phenotype and functionality of MVA-infected DCs. As previously reported, we found that human monocyte-derived DCs upregulated CD86 and HLA-DR in response to MVA infection. Moreover, infected DCs produced a broad array of chemokines and cytokines and were able to activate and induce gamma interferon (IFN-γ) production both in CD4(+) and in CD8(+) allogeneic T cells and in specific autologous peripheral blood lymphocytes (PBLs). Analysis of DC maturation following infection with a recombinant green fluorescent protein (GFP)-expressing MVA revealed that upregulation of CD86 expression was mainly observed in GFP(neg) (bystander) cells. While GFP(pos) (infected) DCs produced tumor necrosis factor alpha (TNF-α), they were unable to produce CXCL10 and were less efficient at inducing IFN-γ production in CEF-specific autologous PBLs. Maturation of bystander DCs could be achieved by incubation with supernatant from infected cultures or with apoptotic infected cells. Type I IFNs were partially responsible for the induction of CXCL10 on bystander DCs. Our findings demonstrate for the first time that, in MVA-infected DC cultures, the leading role with respect to functionality and maturation characteristics is achieved by the bystander DCs. AU - Pascutti, M.F.* AU - Rodríguez, A.M.* AU - Falivene, J.* AU - Giavedoni, L.* AU - Drexler, I. AU - Gherardi, M.M.* C1 - 6399 C2 - 28615 SP - 5532-5545 TI - Interplay between modified vaccinia virus Ankara and dendritic cells: Phenotypic and functional maturation of bystander dendritic cells. JO - J. Virol. VL - 85 IS - 11 PB - Amer Soc Microbiology PY - 2011 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr Virus (EBV) is an ubiquitous human herpesvirus which can lead to infectious mononucleosis and different cancers. In immunocompromised individuals, this virus is a major cause for morbidity and mortality. Transplant patients who did not encounter EBV prior to immunosuppression frequently develop EBV-associated malignancies, but a prophylactic EBV vaccination might reduce this risk considerably. Virus-like particles (VLPs) mimic the structure of the parental virus but lack the viral genome. Therefore, VLPs are considered safe and efficient vaccine candidates. We engineered a dedicated producer cell line for EBV-derived VLPs. This cell line contains a genetically modified EBV genome which is devoid of all potential viral oncogenes but provides viral proteins essential for the assembly and release of VLPs via the endosomal sorting complex required for transport (ESCRT). Human B cells readily take up EBV-based VLPs and present viral epitopes in association with HLA molecules to T cells. Consequently, EBV-based VLPs are highly immunogenic and elicit humoral and strong CD8(+) and CD4(+) T cell responses in vitro and in a preclinical murine model in vivo. Our findings suggest that VLP formulations might be attractive candidates to develop a safe and effective polyvalent vaccine against EBV. AU - Ruiss, R. AU - Jochum, S. AU - Wanner, G.* AU - Reisbach, G. AU - Hammerschmidt, W. AU - Zeidler, R.* C1 - 6361 C2 - 29167 SP - 13105-13113 TI - A virus-like particle-based Epstein-Barr virus vaccine. JO - J. Virol. VL - 85 IS - 24 PB - American Society for Microbiology PY - 2011 SN - 0022-538X ER - TY - JOUR AB - Apoptosis of infected cells is critically involved in antiviral defense. Apoptosis, however, may also support the release and spread of viruses. Although the elimination of infected hepatocytes is required to combat hepatitis B virus (HBV) infection, it is still unknown which consequences hepatocyte apoptosis has for the virus and whether or not it is advantageous to the virus. To study this, we designed a cell culture model consisting of both HBV-producing cell lines and primary human hepatocytes serving as an infection model. We showed that the release of mature, enveloped virions was 80% to 90% reduced 24 h after the induction of apoptosis in HBV-replicating hepatoma cells or HBV-infected hepatocytes. Importantly, HBV particles released from apoptotic hepatocytes were immature and nonenveloped and proved not to be infectious. We found an inverse correlation between the strength of an apoptotic stimulus and the infectivity of the virus particles released: the more potent the apoptotic stimulus, the higher the ratio of nonenveloped capsids to virions and the lower their infectivity. Furthermore, we demonstrated that HBV replication and, particularly, the expression of the HBx protein transcribed from the viral genome during replication do not sensitize cells to apoptosis. Our data clearly reject the hypothesis that the apoptosis of infected hepatocytes facilitates the propagation of HBV. Rather, these data indicate that HBV needs to prevent the apoptosis of its host hepatocyte to ensure the release of infectious progeny and, thus, virus spread in the liver. AU - Arzberger, S. AU - Hösel, M.* AU - Protzer, U. C1 - 4486 C2 - 27779 SP - 11994-12001 TI - Apoptosis of hepatitis B virus-infected hepatocytes prevents release of infectious virus. JO - J. Virol. VL - 84 IS - 22 PB - American Society for Microbiology PY - 2010 SN - 0022-538X ER - TY - JOUR AB - The gp350 glycoprotein encoded by BLLF1 is crucial for efficient Epstein-Barr virus (EBV) infection of resting B cells. Gp350 binds to CD21, but whether this interaction sums up its functions remains unknown. We generated gp350-null EBVs that display CD19-, CD21-, or CD22-specific antibodies at their surface (designated as {Delta}BLLF1-Ab). Gp350-complemented ({Delta}BLLF1-C) and {Delta}BLLF1-Ab were found to bind equally well to B cells. Surprisingly, {Delta}BLLF1 binding was reduced only 1.7-fold relative to its complemented counterparts. Furthermore, B cells exposed to {Delta}BLLF1-Ab or {Delta}BLLF1 viruses presented structural antigens with comparable efficiency and achieved 25 to 80% of the T-cell activation elicited by {Delta}BLLF1-C. These findings show that the gp350-CD21 interaction pair plays only a modest role during virus transfer to the endosomal compartment. However, primary B cells or Raji B cells infected with {Delta}BLLF1-C viruses displayed a 35- to 70-fold higher infection rates than those exposed to {Delta}BLLF1, {Delta}BLLF1-CD22Ab, or {Delta}BLLF1-CD19Ab viruses. Complementation of the gp350 knockout phenotype with CD21Ab substantially enhanced infection rates relative to {Delta}BLLF1 but remained sevenfold (Raji B-cell line) to sixfold (primary B cells) less efficient than with gp350. We therefore infer that gp350 mainly exerts its functions after the internalization step, presumably during release of the viral capsid from the endosomal compartment, and that CD21-dependent but also CD21-independent molecular mechanisms are involved in this process. The latter appear to be characteristic of B-cell infection since transfection of CD21 in 293 cells improved the infection rates with both {Delta}BLLF1-CD21Ab and {Delta}BLLF1-C to a similar extent. AU - Busse, C.* AU - Feederle, R.* AU - Schnölzer, M.* AU - Behrends, U. AU - Mautner, J. AU - Delecluse, H.-J.* C1 - 2951 C2 - 27074 SP - 1139-1147 TI - Epstein-Barr viruses that express a CD21 antibody provide evidence that gp350's functions extend beyond B-cell surface binding. JO - J. Virol. VL - 84 IS - 2 PB - American Society for Microbiology PY - 2010 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus efficiently infects human B cells. The EBV genome is maintained extrachromosomally and replicates synchronously with the host's chromosomes. The latent origin of replication (oriP) guarantees plasmid stability by mediating two basic functions: replication and segregation of the viral genome. While the segregation process of EBV genomes is well understood, little is known about its chromatin association and nuclear distribution during interphase. Here, we analyzed the nuclear localization of EBV genomes and the role of functional oriP domains FR and DS for basic functions such as the transformation of primary cells, their role in targeting EBV genomes to distinct nuclear regions, and their association with epigenetic domains. Fluorescence in situ hybridization visualized the localization of extrachromosomal EBV genomes in the regions adjacent to chromatin-dense territories called the perichromatin. Further, immunofluorescence experiments demonstrated a preference of the viral genome for histone 3 lysine 4-trimethylated (H3K4me3) and histone 3 lysine 9-acetylated (H3K9ac) nuclear regions. To determine the role of FR and DS for establishment and subnuclear localization of EBV genomes, we transformed primary human B lymphocytes with recombinant mini-EBV genomes containing different oriP mutants. The loss of DS results in a slightly increased association in H3K27me3 domains. This study demonstrates that EBV genomes or oriP-based extrachromosomal vector systems are integrated into the higher order nuclear organization. We found that viral genomes are not randomly distributed in the nucleus. FR but not DS is crucial for the localization of EBV in perichromatic regions that are enriched for H3K4me3 and H3K9ac, which are hallmarks of transcriptionally active regions. AU - Deutsch, M.J. AU - Ott, E. AU - Papior, P. AU - Schepers, A. C1 - 423 C2 - 27245 SP - 2533-2546 TI - The latent origin of replication of Epstein-Barr virus directs viral genomes to active regions of the nucleus. JO - J. Virol. VL - 84 IS - 5 PB - American Society for Microbiology PY - 2010 SN - 0022-538X ER - TY - JOUR AB - In cells infected with the Kaposi's sarcoma-associated herpesvirus (KSHV), CSL/CBF1 signaling is essential for viral replication and promotes the survival of KSHV-infected cells. CSL/CBF1 is a DNA adaptor molecule which recruits coactivator and corepressor complexes to regulate viral and cellular gene transcription and which is a major downstream effector molecule of activated Notch. The interaction of KSHV RTA and LANA with CSL/CBF1 has been shown to balance the lytic and latent viral life cycle. Here we report that a third KSHV protein, viral interferon regulatory factor 4 (vIRF4/K10), but none of the three other KSHV-encoded vIRFs, interacts with CSL/CBF1. Two regions of vIRF4 with dissimilar affinities contribute to CSL/CBF1 binding. Similar to Notch, vIRF4 targets the hydrophobic pocket in the beta trefoil domain of CSL/CBF1 through a short peptide motif which closely resembles a motif found in Notch but does not strictly follow the ΦWΦP consensus conserved in human and mouse Notch proteins. Our results suggest that vIRF4 might compete with Notch for CSL/CBF1 binding and signaling. AU - Heinzelmann, K. AU - Scholz, B.A. AU - Nowak, A. AU - Fossum, E.* AU - Kremmer, E. AU - Haas, J.* AU - Frank, R.* AU - Kempkes, B. C1 - 4989 C2 - 27724 SP - 12255-12264 TI - Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 4 (vIRF4/K10) is a novel interaction partner of CSL/CBF1, the major downstream effector of Notch signaling. JO - J. Virol. VL - 84 IS - 23 PB - American Society for Microbiology PY - 2010 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cells in vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection. AU - Iskra, S. AU - Kalla, M. AU - Delecluse, H.J.* AU - Hammerschmidt, W. AU - Moosmann, A. C1 - 5766 C2 - 27727 SP - 3612-3623 TI - Toll-like receptor agonists synergistically increase proliferation and activation of B cells by Epstein-Barr virus. JO - J. Virol. VL - 84 IS - 7 PB - American Society for Microbiology PY - 2010 SN - 0022-538X ER - TY - JOUR AB - Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (gammaHV) infections. To completely explore the potential of the MHV-68 system for the investigation of gammaHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of gammaHV miRNAs. AU - Zhu, J.Y.* AU - Strehle, M.L. AU - Frohn, A.* AU - Kremmer, E. AU - Höfig, K.P. AU - Meister, G.* AU - Adler, H. C1 - 2593 C2 - 27450 SP - 10266-10275 TI - Identification and analysis of expression of novel microRNAs of murine gammaherpesvirus 68. JO - J. Virol. VL - 84 IS - 19 PB - American Society for Microbiology PY - 2010 SN - 0022-538X ER - TY - JOUR AB - Although transcription factors of the basic-helix-loop-helix family have been shown to regulate enhancers of lymphomagenic gammaretroviruses through E-box motifs, overlap of an E-box motif (Egre) with the glucocorticoid response element (GRE) has obscured their function in vivo. We here report that Egre, but not the GRE affects disease induction by the murine T-lymphomagenic SL3-3 virus. Mutating all three copies of Egre prolonged the tumor latency period from 60 to 109 days. Further mutating an E-box motif (Ea/s) outside the enhancer prolonged the latency period to 180 days, suggesting that Ea/s works as a back-up site for Egre. While SL3-3 wt, GRE and Ea/s mutants induced exclusively T-cell lymphomas with wild type latencies, mainly of the CD4+CD8- phenotype, the Egre as well as the Egre plus Ea/s mutants induced B-cell lymphomas and myeloid leukemia in addition to T-cell lymphomas. T-cell lymphomas induced by the two Egre mutants had the same phenotype as those induced by SL3-3 wt, indicating incomplete disruption of T-cell lymphomagenesis in contrast to previous findings for a Runx site mutant of SL3-3. Mutating the Egre site or Egre plus Ea/s triggered several tumor phenotype-associated secondary enhancer changes encompassing neighboring sites, none of which led to regeneration of an E-box motif. Altogether, our results demonstrate a role for the E-box, but not the GRE in T-lymphomagenesis by SL3-3, unveil an inherent broader disease specificity of the virus, and strengthen the notion of selection for more potent enhancer variants of mutated viruses during tumor development. AU - Ejegod, D.* AU - Sørensen, K.D.* AU - Moßbrugger, I. AU - Quintanilla-Martinez, L. AU - Schmidt, J. AU - Pedersen, F.S.* C1 - 4985 C2 - 25755 SP - 336-346 TI - Control of pathogenicity and disease specificity of a T-lymphomagenic gammaretrovirus by E-box motifs but not by an overlapping glucocorticoid response element. JO - J. Virol. VL - 83 IS - 1 PB - ASM PY - 2009 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-kappa B in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-kappa B are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV. AU - Faumont, N.* AU - Durand-Panteix, S.* AU - Schlee, M. AU - Grömminger, S. AU - Schuhmacher, M.* AU - Hölzel, M. AU - Laux, G. AU - Mailhammer, R. AU - Rosenwald, A.* AU - Staudt, L.M.* AU - Bornkamm, G.W. AU - Feuillard, J.* C1 - 1683 C2 - 27014 SP - 5014-5027 TI - C-Myc and Rel/NF-κB are the two master transcriptional systems activated in the latency III program of Epstein-Barr virus-immortalized B cells. JO - J. Virol. VL - 83 IS - 10 PB - Amer Soc Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (gamma HV) infections. According to the colinear organization of the gamma HV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68. AU - Flach, B. AU - Steer, B. AU - Thakur, N.N. AU - Haas, J.* AU - Adler, H. C1 - 1069 C2 - 26298 SP - 8163-8172 TI - The M10 locus of murine gammaherpesvirus 68 contributes to both the lytic and the latent phases of infection. JO - J. Virol. VL - 83 IS - 16 PB - Amer Soc Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5' long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5' LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-D-glucosamine(O-GlcNAc), we evaluated whether increased O-GlcNAc-ylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O- GlcNAcylation-enhancing agent glucosamine ( GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O- GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O- GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4(+) T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (> 95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS. AU - Jochmann, R.* AU - Thurau, M.* AU - Jung, S.* AU - Hofmann, C.* AU - Naschberger, E.* AU - Kremmer, E. AU - Harrer, T.* AU - Miller, M.* AU - Schaft, N.* AU - Stürzl, M.* C1 - 2016 C2 - 26506 SP - 3704-3718 TI - O-linked N-acetylglucosaminylation of Sp1 inhibits the human immunodeficiency virus type 1 promoter. JO - J. Virol. VL - 83 IS - 8 PB - Amer Soc Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48- to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins ( E1B-433R) are sufficient for a productive infection. AU - Kindsmüller, K.* AU - Schreiner, S.* AU - Leinenkugel, F.* AU - Groitl, P.* AU - Kremmer, E. AU - Dobner, T.* C1 - 2018 C2 - 26513 CY - Washington SP - 9045-9056 TI - A 49-kilodalton isoform of the adenovirus type 5 early region 1B 55-kilodalton protein is sufficient to support virus replication. JO - J. Virol. VL - 83 IS - 18 PB - Amer Soc Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4(+) lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector. AU - Lehmann, M.H. AU - Kastenmüller, W. AU - Kandemir, J.D. AU - Brandt, F. AU - Suezer, Y.* AU - Sutter, G.* C1 - 1347 C2 - 26429 SP - 2540-2552 TI - Modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of CCL2 expression. JO - J. Virol. VL - 83 IS - 6 PB - Amer Soc Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - The hepatitis B virus (HBV) core protein (CP) forms the shell of an icosahedral nucleocapsid. In a former work, we identified 11 amino acid residues of CP exposed on the capsid surface by an alanine mutation scan as being important for capsid envelopment. We now introduced several other amino acids at six of these positions and found that almost all 27 tested point mutations at S17, K96, and I126 reproduced the phenotype of the alanine mutation (with only two exceptions): the formation of nucleocapsids and of the viral DNA genome was wild type, but capsid envelopment and virion release were strongly inhibited. This indicates that these side chains have a very specific function during nucleocapsid envelopment. We also identified several CP point mutations (e.g., F122V/S/Y and R127D/G) allowing the formation of capsids but preventing the packaging of pregenomic RNA. The envelopment of such mutant capsids was blocked. Apparently, these CP mutations hampered the recognition/packaging of the pregenome-P-protein complex by CP, a process which is still barely understood, and the mutant capsids devoid of HBV-specific nucleic acid did not express the capsid maturation signal required for envelopment. AU - Pairan, A.* AU - Bruß, V. C1 - 882 C2 - 26579 CY - Washington SP - 11616-11623 TI - Functional surfaces of the hepatitis B virus capsid. JO - J. Virol. VL - 83 IS - 22 PB - American Society for Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction. AU - Schön, U. AU - Diem, O.C. AU - Leitner, L. AU - Günzburg, W.H.* AU - Mager, D.L.* AU - Salmons, B.* AU - Leib-Mösch, C. C1 - 807 C2 - 26619 CY - Washington SP - 12643-12650 TI - Human endogenous retroviral long terminal repeat sequences as cell type-specific promoters in retroviral vectors. JO - J. Virol. VL - 83 IS - 23 PB - American Society for Microbiology PY - 2009 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV)-specific T-cell lines generated by repeated stimulation with EBV-immortalized lymphoblastoid B-cell lines (LCL) have been successfully used to treat EBV-associated posttransplant lymphoproliferative disease (PTLD) in hematopoietic stem cell transplant recipients. However, PTLD in solid-organ transplant recipients and other EBV-associated malignancies respond less efficiently to this adoptive T-cell therapy. LCL-stimulated T-cell preparations are polyclonal and contain CD4(+) and CD8(+) T cells, but the composition varies greatly between lines. Because T-cell lines with higher CD4(+) T-cell proportions show improved clinical efficacy, we assessed which factors might compromise the expansion of this T-cell population. Here we show that spontaneous virus production by LCL and, hence, the presentation of viral antigens varies intra- and interindividually and is further impaired by acyclovir treatment of LCL. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4(+) T cells, whereas human serum from EBV-seropositive donors diminished viral antigen presentation. To overcome these limitations, we used peripheral blood mononuclear cells pulsed with nontransforming virus-like particles as antigen-presenting cells. This strategy facilitated the specific and rapid expansion of EBV-specific CD4(+) T cells and, thus, might contribute to the development of standardized protocols for the generation of T-cell lines with improved clinical efficacy. AU - Adhikary, D. AU - Behrends, U. AU - Feederle, R.* AU - Delecluse, H.J.* AU - Mautner, J. C1 - 136 C2 - 25428 SP - 3903-3911 TI - Standardized and highly efficient expansion of Epstein-Barr virus-specific CD4+ T cells by using virus-like particles. JO - J. Virol. VL - 82 IS - 8 PB - ASM PY - 2008 SN - 0022-538X ER - TY - JOUR AB - Human endogenous retroviruses (HERVs) account for up to 9% of the human genome and include more than 800 elements related to betaretroviruses. While mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice, the role of retroviral elements in human breast cancer remains elusive. Here, we performed a comprehensive microarray-based analysis of overall retroviral transcriptional activities in 46 mammary gland tissue specimens representing pairs of nonmalignant and tumor samples from 23 patients. An analysis of nonmalignant tissue samples revealed a distinct, mammary gland-specific HERV expression profile that consists of 18 constitutively active HERV taxa. For corresponding tumor samples, a general trend toward lower levels of HERV transcription was observed, suggesting common regulatory mechanisms. In various subsets of patients, however, increased transcript levels of single class I HERV families (HERV-T, HERV-E, and HERV-F) and several class II families, including HML-6, were detected. An analysis of transcribed HML-6 sequences revealed either the activation of some or the increased activity of several proviral loci. No evidence for MMTV or human MMTV-like virus transcripts was found, indicating that transcriptionally active, MMTV analogous, exogenous viruses were not present in the breast cancer samples analyzed. AU - Frank, O.* AU - Verbeke, C.* AU - Schwarz, N.* AU - Mayer, J. AU - Fabarius, A.* AU - Hehlmann, R.* AU - Leib-Mösch, C. AU - Seifarth, W.* C1 - 2578 C2 - 25319 SP - 1808-1818 TI - Variable transcriptional activity of endogenous retroviruses in human breast cancer. JO - J. Virol. VL - 82 IS - 4 PB - Amer. Soc. Microbiology PY - 2008 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline double-inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B cells, as shown by annexin V labeling, sub-G(1) peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-kappaB activation induces Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-kappaB, revealing an ambivalent function of LMP1 in cell survival and programmed cell death. AU - le Clorennec, C.* AU - Ouk, TS.* AU - Youlyouz-Marfak, I.* AU - Panteix, S.* AU - Martin, C.C.* AU - Rastelli, J.* AU - Adriaenssens, E.* AU - Zimber-Strobl, U. AU - Coll, J.* AU - Feuillard, J.* AU - Jayat-Vignoles, C.* C1 - 3786 C2 - 25459 SP - 6721-6733 TI - Molecular basis of cytotoxicity of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) in EBV latency III B cells: LMP1 induces type II ligand-independent autoactivation of CD95/Fas with caspase 8-mediated apoptosis. JO - J. Virol. VL - 82 IS - 13 PB - ASM PY - 2008 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research. AU - Lindner, S.E.* AU - Zeller, K. AU - Schepers, A. AU - Sugden, B.* C1 - 4891 C2 - 25415 SP - 5693-5702 TI - The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency. JO - J. Virol. VL - 82 IS - 12 PB - ASM PY - 2008 SN - 0022-538X ER - TY - JOUR AB - Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases. AU - Sander, G.* AU - Konrad, A.* AU - Thurau, M.* AU - Wies, E.* AU - Leubert, R.* AU - Kremmer, E. AU - Dinkel, H.* AU - Schulz, T.* AU - Neipel, F.* AU - Stürzl, M.* C1 - 4225 C2 - 25704 SP - 1908-1922 TI - Intracellular localization map of human herpesvirus 8 proteins. JO - J. Virol. VL - 82 IS - 4 PB - ASM PY - 2008 SN - 0022-538X ER - TY - JOUR AB - A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt. AU - Adler, H. AU - Steer, B. AU - Freimüller, K. AU - Haas, J.* C1 - 5870 C2 - 24488 SP - 7300-7305 TI - Murine Gammaherpesvirus 68 Contains Two Functional Lytic Origins of Replication. JO - J. Virol. VL - 81 IS - 13 PB - ASM PY - 2007 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV)-negative diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma-derived cell lines infected in vitro with a recombinant EBV expressed type II/III latency. High expression of EBNA2 inversely correlated with expression of germinal center (GC)-associated genes, BCL6 and TCL1. The decreased expression of BCL6 appeared to be dose dependent, with almost complete abrogation in highly EBNA2-expressing clones. The role of EBNA2 in negative regulation of these genes was confirmed by transfection and in a hormone-inducible EBNA2 cell system. LMP1 transfection reduced expression of TCL1, but not of BCL6, in DLBCLs. The GC-associated gene repression was at the transcriptional level and CBF1 independent. A decrease in HLA-DR, surface immunoglobulin M, and class II transactivator expression and an increase in CCL3, a BCL6 repression target, was observed in EBNA2-expressing clones. Since BCL6 is indispensable for GC formation and somatic hypermutations (SHM), we suggest that the previously reported lack of SHM seen in EBNA2-expressing GC cells from infectious mononucleosis tonsils could be due to negative regulation of BCL6 by EBNA2. These findings suggest that EBNA2 interferes with the GC phenotype. AU - Boccellato, F.* AU - Anastasiadou, E.* AU - Rosato, P.* AU - Kempkes, B. AU - Frati, L.* AU - Faggioni, A.* AU - Trivedi, P.* C1 - 4649 C2 - 24369 SP - 2274-2282 TI - EBNA2 Interferes with the Germinal Center Phenotype by Downregulating BCL6 and TCL1 in Non-Hodgkin's Lymphoma Cells. JO - J. Virol. VL - 81 IS - 5 PB - ASM PY - 2007 SN - 0022-538X ER - TY - JOUR AB - Recombinant vaccines based on modified vaccinia virus Ankara (MVA) have an excellent record concerning safety and immunogenicity and are currently being evaluated in numerous clinical studies for immunotherapy of infectious diseases and cancer. However, knowledge about the biological properties of target antigens to efficiently induce MVA vaccine-mediated immunity in vivo is sparse. Here, we examined distinct antigen presentation pathways and different antigen formulations contained in MVA vaccines for their capability to induce cytotoxic CD8(+) T-cell (CTL) responses. Strikingly, we found that CTL responses against MVA-produced antigens were dominated by cross-priming in vivo, despite the ability of the virus to efficiently infect professional antigen-presenting cells such as dendritic cells. Moreover, stable mature protein was preferred to preprocessed antigen as the substrate for cross-priming. Our data are essential for improved MVA vaccine design, as they demonstrate the need for optimal adjustment of the target antigen properties to the intrinsic requirements of the delivering vector system. AU - Gasteiger, G. AU - Kastenmüller, W.* AU - Ljapoci, R. AU - Sutter, G.* AU - Drexler, I. C1 - 3325 C2 - 24636 SP - 11925-11936 TI - Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus ankara vector vaccines. JO - J. Virol. VL - 81 IS - 21 PB - ASM PY - 2007 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) can infect various cell types but limits its classical growth-transforming function to B lymphocytes, the cells in which it persists in vivo. Transformation initiates with the activation of Wp, a promoter present as tandemly repeated copies in the viral genome. Assays with short Wp reporter constructs have identified two promoter-activating regions, one of which (UAS2) appears to be lineage independent, while the other (UAS1) was B-cell specific and contained two putative binding sites for the B-cell-specific activator protein BSAP/Pax5. To address the physiologic relevance of these findings, we first used chromosome immunoprecipitation assays and found that BSAP is indeed bound to Wp sequences on the EBV genome in transformed cells. Thereafter, we constructed recombinant EBVs carrying two Wp copies, both wild type, with UAS1 or UAS2 deleted, or mutated in the BSAP binding sites. All the viruses delivered their genomes to the B-cell nucleus equally well. However, the BSAP binding mutant (and the virus with UAS1 deleted) showed no detectable activity in B cells, whether measured by early Wp transcription, expression of EBV latent proteins, or outgrowth of transformed cells. This was a B-cell-specific defect since, on entry into epithelial cells, an environment where Wp is not the latent promoter of choice, all the Wp mutant viruses initiated infection as efficiently as wild-type virus. We infer that EBV ensures the B-cell specificity of its growth-transforming function by exploiting BSAP/Pax5 as a lineage-specific activator of the transforming program. AU - Tierney, R.* AU - Nagra, J.* AU - Hutchings, I. AU - Shannon-Lowe, C.* AU - Altmann, M. AU - Hammerschmidt, W. AU - Rickinson, A.B. AU - Bell, A. C1 - 2211 C2 - 24739 SP - 10092-10100 TI - Epstein-Barr virus exploits BSAP/Pax5 to achieve the B-cell specificity of its growth-transforming program. JO - J. Virol. VL - 81 IS - 18 PB - ASM PY - 2007 SN - 0022-538X ER - TY - JOUR AU - Feederle, R.* AU - Neuhierl, B.* AU - Baldwin, G.* AU - Bannert, H.* AU - Hub, B.* AU - Mautner, J. AU - Behrends, U. AU - Delecluse, H.J.* C1 - 3990 C2 - 24080 SP - 9435-9443 TI - Epstein-barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes. JO - J. Virol. VL - 80 IS - 19 PY - 2006 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) is associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways and contributes to EBV-associated oncogenesis. Regulation of LMP1 expression has been extensively described during the type III latency of EBV. Nevertheless, in the majority of EBV-associated tumors, the virus is commonly found to display a type II latency program in which it is still unknown which viral or cellular protein is really involved in maintaining LMP1 expression. Here, we demonstrate that LMP1 activates its own promoter pLMP1 through the JNK signaling pathway emerging from the TES2 domain. Our results also reveal that this activation is tightly controlled by LMP1, since pLMP1 is inhibited by LMP1-activated NF-κB signaling pathway. By using our physiological models of EBV-infected cells displaying type II latency as well as lymphoblastoid cell lines expressing a type III latency, we also demonstrate that this balanced autoregulation of LMP1 is shared by both latency programs. Finally, we show that this autoactivation is the most important mechanism to maintain LMP1 expression during the type II latency program of EBV. Copyright © 2006, American Society for Microbiology. All Rights Reserved. AU - Goormachtigh, G.* AU - Ouk, T.S.* AU - Mougel, A.* AU - Tranchand-Bunel, D.* AU - Masy, E.* AU - Le, Clorennec, C.* AU - Feuillard, J.* AU - Bornkamm, G.W. AU - Auriault, C.* AU - Manet, E.* AU - Fafeur, V.* AU - Adriaenssens, E.* AU - Coll, J.* C1 - 3556 C2 - 23822 SP - 7382-7393 TI - Autoactivation of the Epstein-barr virus oncogenic protein LMP1 during type II latency through opposite roles of the NF-kB and JNK signaling pathways. JO - J. Virol. VL - 80 IS - 15 PY - 2006 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines. Copyright © 2006, American Society for Microbiology. All Rights Reserved. AU - Maier, S. AU - Staffler, G. AU - Hartmann, A. AU - Höck, J. AU - Henning, K.* AU - Grabusic, K. AU - Mailhammer, R. AU - Hoffmann, R.* AU - Wilmanns, M.* AU - Lang, R.* AU - Mages, J.* AU - Kempkes, B. C1 - 3557 C2 - 23823 SP - 9761-9771 TI - Cellular target genes of Epstein-Barr virus nuclear antigen 2. JO - J. Virol. VL - 80 IS - 19 PY - 2006 SN - 0022-538X ER - TY - JOUR AU - Milosevic, S. AU - Behrends, U. AU - Adhikary, D. AU - Mautner, J. C1 - 3989 C2 - 24079 SP - 10357-10364 TI - Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-barr virus by a novel bacterial expression cloning approach. JO - J. Virol. VL - 80 IS - 21 PY - 2006 SN - 0022-538X ER - TY - JOUR AB - The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-κB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004), Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1 EBNA2 irons-activation of bfl-1 requires CBF1 (or RBP-Jκ), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter, trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-κB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process. Copyright © 2006, American Society for Microbiology. All Rights Reserved. AU - Pegman, P.M.* AU - Smith, S.M.* AU - D'Souza, B.N.* AU - Loughran, S.T.* AU - Maier, S. AU - Kempkes, B. AU - Cahill, P.A.* AU - Simmons, M.J.* AU - Gelinas, C.* AU - Walls, D.* C1 - 4910 C2 - 24025 SP - 8133-8144 TI - Epstein-Barr virus nuclear antigen 2 trans-activates the cellular antiapoptotic bfl-1 gene by a CBF1/RBPJk-dependent pathway. JO - J. Virol. VL - 80 IS - 16 PY - 2006 SN - 0022-538X ER - TY - JOUR AU - Stengel, A. AU - Roos, C.* AU - Hunsmann, G.* AU - Seifarth, W.* AU - Leib-Mösch, C. AU - Greenwood, A.D. C1 - 2889 C2 - 23517 SP - 4415-4421 TI - Expression profiles of endogenous in Old World Monkeys. JO - J. Virol. VL - 80 IS - 9 PY - 2006 SN - 0022-538X ER - TY - JOUR AB - Human endogenous retroviruses (HERVs) are a major component of the human genome and an active part of the transcriptome. Some HERVs play vital biological roles, while others potentially contribute to diseases. Many HERVs are relatively new in the primate genome, having entered or expanded after the lineages leading to the platyrrhines (New World monkeys) and catarrhines (Old World monkeys and apes) separated. Most HERVs are active in at least some tissues, though tissue specificity is common for most elements. We analyzed multiple tissues from several Old World monkeys using retroviral pol-based DNA microarrays and quantitative PCR methods to determine their ERV expression profiles. The results demonstrate that while many ERVs are active in nonhuman primates, overall the tissue expression specificity is unique to each species. Most striking is that while the majority of HERVs analyzed in this study are expressed in human brain, almost none are expressed in Old World monkey brains or are only weakly expressed.Α AU - Stengel, A. AU - Roos, C.* AU - Hunsmann, G.* AU - Seifarth, W.* AU - Leib-Mösch, C. AU - Greenwood, A.D. C1 - 8692 C2 - 29444 SP - 4415-4421 TI - Expression profiles of endogenous retroviruses in Old World monkeys. JO - J. Virol. VL - 80 IS - 9 PB - American Society for Microbiology PY - 2006 SN - 0022-538X ER - TY - JOUR AB - The detection and identification of retroviral transcripts in brain samples, cerebrospinal fluid, and plasma of individuals with recent-onset schizophrenia and schizoaffective disorders suggest that activation or upregulation of distinct human endogenous retroviruses (HERVs) may play a role in the etiopathogenesis of neuropsychiatric diseases. To test this hypothesis, we performed a comprehensive microarray-based analysis of HERV transcriptional activity in human brains. We investigated 50 representative members of 20 HERV families in a total of 215 brain samples derived from individuals with schizophrenia or bipolar disorders and matched controls. A characteristic brain-specific retroviral activity profile was found that consists of members of the class I families HERV-E, HERV-F, and ERV9 and members of HERV-K taxa. In addition to these constitutively expressed HERVs, a number of differentially active HERV elements were identified in all brain samples independent of the disease pattern that may reflect differences in the genetic background of the tested individuals. Only a subgroup of the HML-2 family (HERV-K10) was significantly overrepresented in both bipolar-disorder- and schizophrenia- associated samples compared to healthy brains, suggesting a potential association with disease. Real-time PCR analysis of HERV env transcripts with coding capacity potentially involved in neuroinflammatory conditions revealed that env expression of HERV-W, HERV-FRD, and HML-2 remains unaffected regardless of the clinical picture. Our data suggest that HERV transcription in brains is weakly correlated with schizophrenia and related diseases but may be influenced by the individual genetic background, brain-infiltrating immune cells, or medical treatment. Copyright © 2005, American Society for Microbiology. All Rights Reserved. AU - Frank, O.* AU - Giehl, M.* AU - Zheng, C.* AU - Hehlmann, R.* AU - Leib-Mösch, C. AU - Seifarth, W.* C1 - 3022 C2 - 22845 SP - 10890-10901 TI - Human endogenous retrovirus expression profiles in samples from brains of patients with schizophrenia and bipolar disorders. JO - J. Virol. VL - 79 IS - 17 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - Most Epstein-Barr virus (EBV)-positive Burkitt's lymphomas (BLs) carry a wild-type EBV genome and express EBV nuclear antigen 1 (EBNA1) selectively from the BamHI Q promoter (latency I). Recently we identified a distinct subset of BLs carrying both wild-type and EBNA2 gene-deleted (transformation-defective) viral genomes. The cells displayed an atypical "BamHI W promoter (Wp)-restricted" form of latency where Wp (rather than Qp) was active and EBNA1, -3A, -3B, -3C, and -LP were expressed in the absence of EBNA2 or latent membrane proteins 1 and 2. Here we present data strongly supporting the view that the EBNA2-deleted genome is transcriptionally active in these cells and the wild-type genome is silent. Single-cell cloning of three parental Wp-restricted BL lines generated clones carrying either both viral genomes or the EBNA2-deleted genome only, never clones with the wild-type genome only. All rescued clones displayed the Wp-restricted form of latency characteristic of the parent line and retained the original parent cell phenotype. Interestingly, Wp-restricted parent lines and derived clones were markedly more resistant to inducers of apoptosis than standard latency I BL lines. Furthermore, in vitro infection of EBV-negative BL lines with an EBNA2 gene-deleted virus generated EBV-positive converts with Wp-restricted latency and a similarly marked apoptosis resistance. We postulate that, in the subset of BLs displaying Wp-restricted latency, infection of a tumor progenitor cell with an EBNA2 gene-deleted virus has provided that cell with a survival advantage through broadening antigen expression to include the EBNA3 proteins. Copyright © 2005, American Society for Microbiology. All Rights Reserved. AU - Kelly, G.K.* AU - Milner, A.E.* AU - Tierney, R.J.* AU - Croom-Carter, D.S.G.* AU - Altmann, M. AU - Hammerschmidt, W. AU - Bell, A.I.* AU - Rickinson, A.B.* C1 - 5407 C2 - 22979 SP - 10709-10717 TI - Epstein-barr virus nuclear antigen 2 (EBNA2) gene deletion is consistently linked with EBNA3A,-3B and -3C expression in burkitt's lymphoma cells and with increased resistance to apoptosis. JO - J. Virol. VL - 79 IS - 16 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-DeltaE3L, was found defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2'-5'-oligoadenylate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-DeltaE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-DeltaE3L infection. Interestingly, a cluster of genes up regulated exclusively in MVA-DeltaE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible protein beta, and dual-specificity protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle. AU - Ludwig, H.* AU - Mages, J.* AU - Staib, C. AU - Lehmann, M.H. AU - Lang, R.* AU - Sutter, G. C1 - 1994 C2 - 22811 SP - 2584-2596 TI - Role of viral factor E3L in modified vaccinia virus Ankara infection of human HeLa cells: Regulation of the virus life cycle and identification of differentially expressed host genes. JO - J. Virol. VL - 79 IS - 4 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies. AU - Maier, S. AU - Santak, M. AU - Mantik, A. AU - Grabusic, K. AU - Kremmer, E. AU - Hammerschmidt, W. AU - Kempkes, B. C1 - 2328 C2 - 22727 SP - 8784-8792 TI - A somatic knockout of CBF1 in a human B-cell line reveals that induction of CD21 and CCR7 by EBNA-2 is strictly CBF1 dependent and that downregulation of immunoglobulin M is partially CBF1 independent. JO - J. Virol. VL - 79 IS - 14 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - Constitutive activation of signal transducer and activator of transcription 1 (STAT1) is a distinctive feature of Epstein-Barr virus (EBV)-immortalized B cells (lymphoblastoid cell lines [LCLs]). The expression of STAT1 in these cells is modulated by the latent membrane protein 1 (LMP1), but the mechanism of STAT1 activation has remained unclear. We demonstrate that the tyrosine phosphorylation of STAT1 in LCLs results from an indirect pathway encompassing an NF-kappaB-dependent secretion of interferons (IFNs). The cell culture supernatant of LCLs induced tyrosine phosphorylation of STAT1 in cells with no constitutively activated STAT1. Moreover, removal of supernatant from LCLs was sufficient to decrease the phosphorylation of STAT1. Inhibition of NF-kappaB activity by different pharmacological inhibitors (i.e., parthenolide, MG132 and BAY 11-7082) and by overexpressed mutated IkappaBalpha prevented the activation of STAT1. To identify the factors involved, we performed macroarray cDNA profiling with or without inhibition of NF-kappaB. The expression of several cytokines was NF-kappaB dependent among those alpha and gamma IFNs (IFN-alpha and IFN-gamma), known activators of STAT1. By real-time PCR and enzyme-linked immunosorbent assay we show that IFN-alpha and IFN-gamma are expressed and released by LCLs in an NF-kappaB-dependent manner. Finally, the blocking of the IFN-alpha and IFN-gamma by neutralizing antibodies led to the complete inhibition of tyrosine phosphorylation of STAT1. Taken together, our results clearly show that LMP1-induced tyrosine phosphorylation of STAT1 is almost exclusively due to the NF-kappaB-dependent secretion of IFNs. Whether this response, which is usually considered to be antiviral, is in fact required for the persistence of the virus remains to be elucidated. AU - Najjar, I.* AU - Schlee, M. AU - Bornkamm, G.W. C1 - 1930 C2 - 22600 SP - 4936-4943 TI - Latent membrane protein 1 regulates STAT1 through NF-kB-dependent interferon secretion in Epstein-Barr virus-immortalized B cells. JO - J. Virol. VL - 79 IS - 8 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - The murine leukemia retrovirus SL3-3 induces lymphomas in the T-cell compartment of the hematopoetic system when it is injected into newborn mice of susceptible strains. Previously, our laboratory reported on a deletion mutant of SL3-3 that induces T-cell tumors faster than the wild-type virus (S. Ethelberg, A. B. Sorensen, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:9796-9799, 1997). PCR analyses of proviral integrations in the promoter region of the c-myc proto-oncogene in lymphomas induced by wild-type SL3-3 [SL3-3(wt)] and the enhancer deletion mutant displayed a difference in targeting frequency into this locus. We here report on patterns of proviral insertions into the c-myc promoter region from SL3-3(wt), the faster variant, as well as other enhancer variants from a total of approximately 250 tumors. The analysis reveals (i) several integration site hot spots in the c-myc promoter region, (ii) differences in integration patterns between SL3-3(wt) and enhancer deletion mutant viruses, (iii) a correlation between tumor latency and the number of proviral insertions into the c-myc promoter, and (iv) a [5'-(A/C/G)TA(C/G/T)-3'] integration site consensus sequence. Unexpectedly, about 12% of the sequenced insertions were associated with point mutations in the direct repeat flanking the provirus. Based on these results, we propose a model for error-prone gap repair of host-provirus junctions. AU - Nielsen, A.A.* AU - Sorensen, A.B.* AU - Schmidt, J. AU - Pedersen, F.S.* C1 - 3616 C2 - 23189 SP - 67-78 TI - Analysis of wild-type and mutant SL3-3 murine leukemia virus insertions in the c-myc promoter during lymphomaganesis reveals target site hot spots, virus-dependent patterns, and frequent error-prone gap repair. JO - J. Virol. VL - 79 IS - 1 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - The mechanistic contribution of the Epstein-Barr virus (EBV) EBNA-LP protein to B-cell immortalization remains an enigma. However, previous studies have indicated that EBNA-LP may contribute to immortalization by enhancing EBNA2-mediated transcriptional activation of the LMP-1 gene. To gain further insight into the potential role EBNA-LP has in EBV-mediated B-cell immortalization, we asked whether it is a global or gene-specific coactivator of EBNA2 and whether coactivation requires interaction between these proteins. In type I Burkitt's lymphoma cells, we found that EBNA-LP strongly coactivated EBNA2 stimulation of LMP-1 and LMP2B RNAs, which are expressed from the viral divergent promoter. Surprisingly, the viral LMP2A gene and cellular CD21 and Hes-1 genes were induced by EBNA2 but showed no further induction after EBNA-LP coexpression. We also found that EBNA-LP did not stably interact with EBNA2 in coimmunoprecipitation assays, even though the conditions were adequate to observe specific interactions between EBNA2 and its cellular cofactor, CBF1. Colocalization between EBNA2 and EBNA-LP was not detectable in EBV-transformed cell lines or transfected type I Burkitt's cells. Finally, no significant interactions between EBNA2 and EBNA-LP were found with mammalian two-hybrid assays. From this data, we conclude that EBNA-LP is not a global coactivator of EBNA2 targets, but it preferentially coactivates EBNA2 stimulation of the viral divergent promoter. While this may require specific transient interactions between these proteins that only occur in the context of the divergent promoter, our data strongly suggest that EBNA-LP also cooperates with EBNA2 through mechanisms that do not require direct or indirect complex formation between these proteins. AU - Peng, R.* AU - Moses, S.C.* AU - Tan, J.* AU - Kremmer, E. AU - Ling, P.D.* C1 - 1282 C2 - 23021 SP - 4492-4505 TI - The Epstein-Barr virus EBNA-LP protein preferentially coactivates EBNA2-mediated stimulation of latent membrane proteins expressed from the viral divergent promoter. JO - J. Virol. VL - 79 IS - 7 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - Retrovirus-like sequences account for 8 to 9% of the human genome. Among these sequences, about 8,000 pol-containing proviral elements have been identified to date. As part of our ongoing search for active and possibly disease-relevant human endogenous retroviruses (HERVs), we have recently developed an oligonucleotide-based microarray. The assay allows for both the detection and the identification of most known retroviral reverse transcriptase (RT)-related nucleic acids in biological samples. In the present study, we have investigated the transcriptional activity of representative members of 20 HERV families in 19 different normal human tissues. Qualitative evaluation of chip hybridization signals and quantitative analysis by real-time RT-PCR revealed distinct HERV activity in the human tissues under investigation, suggesting that HERV elements are active in human cells in a tissue-specific manner. Most active members of HERV families were found in mRNA prepared from skin, thyroid gland, placenta, and tissues of reproductive organs. In contrast, only few active HERVs were detectable in muscle cells. Human tissues that lack HERV transcription could not be found, confirming that human endogenous retroviruses are permanent components of the human transcriptome. Distinct activity patterns may reflect the characteristics of the regulatory machinery in these cells, e.g., cell type-dependent occurrence of transcriptional regulatory factors. AU - Seifarth, W.* AU - Frank, O.* AU - Zeilfelder, U.* AU - Spiess, B.* AU - Greenwood, A.D.* AU - Hehlmann, R.* AU - Leib-Mösch, C. C1 - 2669 C2 - 22480 SP - 341-352 TI - Comprehensive analysis of human endogenous retrovirus transcriptional activity in human tissues with a retrovirus-specific microarray. JO - J. Virol. VL - 79 IS - 1 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol. 75:4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected Xenopus laevis oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s). AU - Smulevitch, S.* AU - Michalowski, D.* AU - Zolutukhin, A.S.* AU - Schneider, R. AU - Bear, J.* AU - Roth, P.* AU - Pavlakis, G.N.* AU - Felber, B.K.* C1 - 5291 C2 - 22476 SP - 2356-2365 TI - Structural and functional analysis of the RNA transport element, a member of an extensive family present in the mouse genome. JO - J. Virol. VL - 79 IS - 4 PY - 2005 SN - 0022-538X ER - TY - JOUR AB - Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2), a direct transcriptional activator of viral and cellular genes, is required for EBV-induced B-cell transformation. The functional role of conserved regions within the amino terminus of the protein preceding the poly-proline region has yet to be fully characterized. Thus, we tested whether the EBNA2 amino-terminal 30 amino acid residues, containing evolutionarily conserved region 1, are required for stimulating viral and cellular gene expression necessary for B-cell transformation in a viral transcomplementation assay. We found that these residues are required for its ability to induce LMP-1 expression in lymphoblastoid cell lines (LCLs), to stimulate LMP-1 promoter reporter plasmids in transient-cotransfection assays, and to rescue LCL growth following inactivation of endogenous wild-type EBNA2 protein. Deletion of amino acid residues 3 to 30 also impaired its ability to self-associate in coimmunoprecipitation assays. These data indicate that EBNA2 residues 3 to 30 comprise an essential domain required for induction of LMP-1 expression and, consequently, for maintenance of the immortalized phenotype of LCLs. The ability to self-associate into dimers or multimers conferred by this domain may be an important mechanism for these effects. AU - Gordadze, A.V.* AU - Onunwor, C.W.* AU - Peng, R.S.* AU - Poston, D.* AU - Kremmer, E. AU - Ling, P.D.* C1 - 1733 C2 - 22106 SP - 3919-3929 TI - EBNA2 amino acids 3 to 30 are required for induction of LMP-1 and immortalization maintenance. JO - J. Virol. VL - 78 IS - 8 PY - 2004 SN - 0022-538X ER - TY - JOUR AB - In addition to functioning as a transcriptional transactivator, Epstein-Barr virus EBNA2 interacts with Nur77 to protect against Nur77-mediated apoptosis. Estrogen-regulated EBNA2 in EREB2-5 cells was replaced by either EBNA2 or EBNA2 with a deletion of conserved region 4 (EBNA2DeltaCR4). Both EBNA2-converted and EBNA2DeltaCR4-converted EREB2-5 cells grew in the absence of estrogen and expressed LMP1. Treatment with tumor necrosis factor alpha did not induce apoptosis of EBNA2- or EBNA2DeltaCR4-expressing cells, but EBNA2DeltaCR4 cells were susceptible to etoposide and 5-fluorouracil, Nur77-mediated inducers of apoptosis. Thus, EBNA2 protects B cells against specific apoptotic agents against which LMP1 is not effective. AU - Lee, J.M.* AU - Lee, K.-H.* AU - Farrell, C.J.* AU - Ling, P.D.* AU - Kempkes, B. AU - Park, J.H.* AU - Hayward, S.D.* C1 - 2795 C2 - 22351 SP - 12694-12697 TI - EBNA2 is required for protection of latently Epstein-Barr virus-infected B cells against specific apoptotic stimuli. JO - J. Virol. VL - 78 IS - 22 PY - 2004 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxy-terminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication. AU - Ruvolo, V.* AU - Sun, L.* AU - Howard, K.* AU - Sung, S.* AU - Delecluse, H.-J.* AU - Hammerschmidt, W. AU - Swaminathan, S.* C1 - 2166 C2 - 21670 SP - 2-13 TI - Functional analysis of Epstein-Barr virus SM protein: Identification of amino acids essential for structure, transactivation, splicing inhibition and virion production. JO - J. Virol. VL - 78 IS - 1 PB - ASM PY - 2004 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitt's lymphoma and Hodgkin's disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization. AU - Schlee, M. AU - Krug, T.* AU - Gires, O. AU - Zeidler, R.* AU - Hammerschmidt, W. AU - Mailhammer, R. AU - Laux, G. AU - Sauer, G.* AU - Lovric, J.* AU - Bornkamm, G.W. C1 - 5055 C2 - 21804 SP - 3941-3952 TI - Identification of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) target proteins by proteome analysis: Activation of EBNA2 in conditionally immortalized B cells reflects early events after infection of primary b cells by EBV. JO - J. Virol. VL - 78 IS - 8 PY - 2004 SN - 0022-538X ER - TY - JOUR AB - The human gammaherpesviruses Epstein-Barr virus and Kaposi Sarcoma-associated herpesvirus both contain a glycoprotein (gp350/220 and K8.1, respectively) that mediates binding to target cells and has been studied in great detail in vitro. However, there is no direct information on the role that these glycoproteins play in pathogenesis in vivo. Infection of mice by murid herpesvirus 4 strain 68 (MHV-68) is an established animal model for gammaherpesvirus pathogenesis and expresses an analogous glycoprotein, gp150. To elucidate the in vivo function of gp150, a recombinant MHV-68 deficient in gp150 production was generated (vgp150Delta). The productive viral replication in vitro and in vivo was largely unaffected by mutation of gp150, aside from a partial defect in the release of extracellular virus. Likewise, B-cell latency was established. However, the transient mononucleosis and spike in latently infected cells associated with the spread of MHV-68 to the spleen was significantly reduced in vgp150Delta-infected mice. A soluble, recombinant gp150 was found to bind specifically to B cells but not to epithelial cells in culture. In addition, gp150-deficient MHV-68 derived from mouse lungs bound less well to spleen cells than wild-type virus. Thus, gp150 is highly similar in function in vitro to the Epstein-Barr virus gp350/220. These results suggest a role for these analogous proteins in mononucleosis and have implications for their use as vaccine antigens. AU - Stewart, J.P.* AU - Silvia, O.J.* AU - Atkin, I.M.D.* AU - Hughes, D.J.* AU - Ebrahimi, B.* AU - Adler, H. C1 - 4991 C2 - 21994 SP - 10449-10459 TI - In vivo function of a gammaherpesvirus virion glycoprotein: Influence on B-cell infection and mononucleosis. JO - J. Virol. VL - 78 IS - 19 PY - 2004 SN - 0022-538X ER - TY - JOUR AB - SL3-3 murine leukemia virus is a potent inducer of T-lymphomas in mice. Using inbred NMRI mice, it was previously reported that a mutant of SL3-3 with all enhancer Runx (AML1/core) sites disrupted by 3-bp mutations (SL3-3dm) induces predominantly non-T-cell tumors with severely extended latency (S. Ethelberg, J. Lovmand, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:7273-7280, 1997). By use of three-color flow cytometry and molecular and histopathological analyses, we have now performed a detailed phenotypic characterization of SL3-3- and SL3-3dm-induced tumors in this mouse strain. All wild-type induced tumors had clonal T-cell receptor beta rearrangements, and the vast majority were CD3(+) CD4(+) CD8(-) T-lymphomas. Such a consistent phenotypic pattern is unusual for murine leukemia virus-induced T-lymphomas. The mutant virus induced malignancies of four distinct hematopoietic lineages: myeloid, T lymphoid, B lymphoid, and erythroid. The most common disease was myeloid leukemia with maturation. Thus, mutation of all Runx motifs in the enhancer of SL3-3 severely impedes viral T-lymphomagenicity and thereby discloses a considerable and formerly unappreciated potential of this virus for myeloid leukemia induction. Proviral enhancers with complex structural alterations (deletions, insertions, and/or duplications) were found in most SL3-3dm-induced T-lymphoid tumors and immature myeloid leukemias but not in any cases of myeloid leukemia with maturation, mature B-lymphoma, or erythroleukemia. Altogether, our results indicate that the SL3-3dm enhancer in itself promotes induction of myeloid leukemia with maturation but that structural changes may arise in vivo and redirect viral disease specificity to induction of T-lymphoid or immature myeloid leukemias, which typically develop with moderately shorter latencies. AU - Sørensen, K.D.* AU - Quintanilla-Martinez, L. AU - Kunder, S. AU - Schmidt, J. AU - Pedersen, F.S.* C1 - 2918 C2 - 22131 SP - 13216-13231 TI - Mutation of all runx (AML1/Core) sites in the enhancer of T-lymphomagenic SL3-3 murine leukemia virus unmasks a significant potential for myeloid leukemia induction and favors enhancer evolution toward induction of other disease patterns. JO - J. Virol. VL - 78 IS - 23 PY - 2004 SN - 0022-538X ER - TY - JOUR AU - Brinkmann, M.M.* AU - Glenn, M.* AU - Rainbow, L.* AU - Kieser, A. AU - Henke-Gendo, C.* AU - Schulz, T.H.* C1 - 22337 C2 - 21194 SP - 9346-9358 TI - Activation of Mitogen-Activated Protein Kinase and NF-kB Pathways by a Kaposi's Sarcoma- Associated Herpesvirus K15 Membrane Protein. JO - J. Virol. VL - 77 PY - 2003 SN - 0022-538X ER - TY - JOUR AU - Dudziak, D. AU - Kieser, A. AU - Dirmeier, U. AU - Nimmerjahn, F.* AU - Berchtold, S.* AU - Steinkasserer, A.* AU - Marschall, G.* AU - Hammerschmidt, W. AU - Laux, G. AU - Bornkamm, G.W. C1 - 22285 C2 - 21075 SP - 8290-8298 TI - Latent Membrane Protein 1 of Epstein-Barr Virus Induces CD83 by the NF-kB Signaling Pathway. JO - J. Virol. VL - 77 PY - 2003 SN - 0022-538X ER - TY - JOUR AU - Herzer, K.* AU - Falk, C.S. AU - Encke, J.* AU - Eichhorst, S.T.* AU - Ulsenheimer, A.* AU - Seliger, B.* AU - Krammer, P.H.* C1 - 22283 C2 - 21067 SP - 8299-8309 TI - Upregulation of Major Histocompatibility Complex Class I on Liver Cells by Hepatitis C Virus Core Protein via p53 and TAP1 Impairs Natural Killer Cell Cytotoxicity. JO - J. Virol. VL - 77 PY - 2003 SN - 0022-538X ER - TY - JOUR AB - Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-DeltaE3L. Surprisingly, we found that MVA-DeltaE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-DeltaE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-alpha/beta) in supernatants of MVA-DeltaE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-alpha inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction. AU - Hornemann, S. AU - Harlin, O.* AU - Staib, C. AU - Kisling, S. AU - Erfle, V. AU - Kaspers, B.* AU - Häcker, G.* AU - Sutter, G. C1 - 22256 C2 - 21022 SP - 8394-8407 TI - Replication of Modified Vaccinia Virus Ankara in Primary Chicken Embryo Fibroblasts Requires Expression of the Interferon Resistance Gene E3L. JO - J. Virol. VL - 77 PY - 2003 SN - 0022-538X ER - TY - JOUR AB - We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1(165-173) peptide for ex vivo restimulation of splenocytes prior to analysis ((51)Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D(b)-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and (51)Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1 Delta N10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and (51)Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins. AU - Öhlschläger, P.* AU - Osen, W.* AU - Dell, K.* AU - Faath, S.* AU - Garcea, R.L.* AU - Jochmus, I.* AU - Müller, M.* AU - Pawlita, M.* AU - Schäfer, K.* AU - Sehr, P.* AU - Staib, C. AU - Sutter, G. AU - Gissmann, L.* C1 - 10025 C2 - 21030 SP - 4635-4645 TI - Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. JO - J. Virol. VL - 77 IS - 8 PY - 2003 SN - 0022-538X ER - TY - JOUR AB - Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte (CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently, SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01(+) macaques with constructs encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-specific CTL and CD4(+) helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL. At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01(+) controls were challenged intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic-phase viral replication after a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus. AU - Vogel, T.U.* AU - Reynolds, M.R.* AU - Fuller, D.H.* AU - Vielhuber, K.* AU - Shipley, T.* AU - Fuller, J.T.* AU - Kunstman, K.J.* AU - Sutter, G. AU - Marthas, M.L.* AU - Erfle, V. AU - Wolinsky, S.M.* AU - Wang, C.* AU - Allison, D.B.* AU - Rud, E.W.* AU - Wilson, N.* AU - Montefiori, D.* AU - Altman, J.D.* AU - Watkins, D.I.* C1 - 23872 C2 - 31369 SP - 13348-13360 TI - Multispecific vaccine-induced mucosal cytotoxic T lymphocytes reduce acute-phase viral replication but fail in long-term control of simian immunodeficiency virus SIVmac239. JO - J. Virol. VL - 77 IS - 24 PB - Amer. Soc. Microbiology PY - 2003 SN - 0022-538X ER - TY - JOUR AU - Allen, T.M.* AU - Mortara, L.* AU - Mothe, B.R.* AU - Liebl, M.* AU - Jing, P.* AU - Calore, B.* AU - Piekarczyk, M.* AU - Rudersdorf, R.* AU - O'Connor, D.H.* AU - Wang, X.* AU - Wang, C.* AU - Allison, D.B.* AU - Altman, J.D.* AU - Sette, A.* AU - Desrosiers, R.C.* AU - Sutter, G. AU - Watkins, D.I.* C1 - 21993 C2 - 20524 SP - 4108-4112 TI - Tat-Vaccinated Macaques Do Not Control Simian Immunodeficiency Virus SIVmac239 Replication. JO - J. Virol. VL - 76 PY - 2002 SN - 0022-538X ER - TY - JOUR AU - Gruffat, H.* AU - Batisse, J.* AU - Pich, D. AU - Neuhierl, W. AU - Manet, E.* AU - Hammerschmidt, W. AU - Sergeant, A.* C1 - 21962 C2 - 20484 SP - 9635-9644 TI - Epstein-Barr Virus mRNA Export Factor EB2 is Essential for Production of Infectious Virus. JO - J. Virol. VL - 76 PY - 2002 SN - 0022-538X ER - TY - JOUR AU - Guérin, J.-L.* AU - Gelfi, J.* AU - Boullier, S.* AU - Delverdier, M.* AU - Bellanger, F.-A.* AU - Bertagnoli, S.* AU - Drexler, I. AU - Sutter, G. AU - Messud-Petit, F.* C1 - 22013 C2 - 20561 SP - 2912-2923 TI - Myxoma Virus Leukemia-Associated Protein is Responsible for Major Histocompatibility Complex Class I and Fas-CD95 Down-Regulation and Defines Scrapins, a New Group of Surface Cellular Receptor Abductor Proteins. JO - J. Virol. VL - 76 PY - 2002 SN - 0022-538X ER - TY - JOUR AB - Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease. AU - Horton, H.* AU - Vogel, T.U.* AU - Carter, D.K.* AU - Vielhuber, K.* AU - Fuller, D.H.* AU - Shipley, T.* AU - Fuller, J.T.* AU - Kunstmann, K.J.* AU - Sutter, G. AU - Montefiori, D.C.* AU - Erfle, V. AU - Desrosiers, R.C.* AU - Wilson, N.* C1 - 10024 C2 - 20523 SP - 7187-7202 TI - Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239. JO - J. Virol. VL - 76 IS - 14 PY - 2002 SN - 0022-538X ER - TY - JOUR AU - Ried, M.U.* AU - Girod, A.* AU - Leike, K.* AU - Buning, H.* AU - Hallek, M. C1 - 22015 C2 - 20566 SP - 4559-4566 TI - Adeno-Associated Virus Capsids Displaying Immunoglobulin-Binding Domains Permit Antibody- Mediated Vector Retargeting to Specific Cell Surface Receptors. JO - J. Virol. VL - 76 PY - 2002 SN - 0022-538X ER - TY - JOUR AB - Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from Xenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism. AU - Nappi, F.* AU - Schneider, R. AU - Zolutukhin, A.* AU - Smulevitch, S.* AU - Michalowski, D.* AU - Bear, J.* AU - Felber, B.K.* AU - Pavlakis, G.N.* C1 - 362 C2 - 22806 SP - 4558-4569 TI - Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap. JO - J. Virol. VL - 75 IS - 10 PY - 2001 SN - 0022-538X ER - TY - JOUR AB - Human herpesvirus 8 (HHV-8) is found in immunoblastic B cells of patients with multicentric Castleman's disease (MCD) and, predominantly in a latent form, in primary effusion lymphoma (PEL) cells and Kaposi's sarcoma (KS) spindle cells. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. One of these viral factors is encoded by open reading frame (ORF) K3. Here we show that in PEL cells, ORF K3 is expressed through viral transcripts that are induced very early upon virus reactivation, including bicistronic RNA molecules containing coding sequences from viral ORFs K3 and 70. Specifically, we found that a bicistronic transcript was expressed in the absence of de novo protein synthesis, thereby identifying a novel HHV-8 immediate-early gene product. Several features of the RNA molecules encoding the K3 product, including multiple transcriptional start sites, multiple donor splicing sites, and potential alternative ATG usage, suggest that there exists a finely tuned modulation of ORF K3 expression. By contrast, ORF K3 transcripts are not detected in the majority of cells present in KS lesions that are latently infected by the virus, suggesting that there are other, as-yet-unknown mechanisms of immune evasion for infected KS spindle cells. Nevertheless, because HHV-8 viremia precedes the development of KS lesions and is associated with the recrudescence of MCD symptoms, the prompt expression of ORF K3 in productively infected circulating cells may be important for virus pathogenesis. Thus, molecules targeting host or viral factors that activate ORF K3 expression or inactivate the biological functions of the K3 product should be exploited for the prevention or treatment of HHV-8-associated diseases in at-risk individuals. AU - Rimessi, P.* AU - Bonaccorsi, A.* AU - Stürzl, M. AU - Fabris, M.* AU - Brocca-Cofano, E.* AU - Caputo, A.* AU - Melucci-Vigo, G.* AU - Falchi, M.* AU - Cafaro, A.* AU - Cassai, E.* AU - Ensoli, B.* AU - Monini, P.* C1 - 21712 C2 - 19905 SP - 7161-7174 TI - Transcription Pattern of Human Herpesvirus 8 Open Reading Frame K3 in Primary Effusion Lymphoma and KaposiS Sarcoma. JO - J. Virol. VL - 75 IS - 15 PY - 2001 SN - 0022-538X ER - TY - JOUR AB - The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells. AU - Janz, A. AU - Oezel, M.* AU - Kurzeder, C. AU - Mautner, J. AU - Pich, D. AU - Kost, M. AU - Hammerschmidt, W. AU - Delecluse, H.-J. C1 - 10023 C2 - 22375 SP - 10142-10152 TI - Infectious Epstein-Barr virus lacking major glycoprotein BLLF1 (gp350/220) demonstrates the existence of additional viral ligands. JO - J. Virol. VL - 74 IS - 21 PY - 2000 SN - 0022-538X ER - TY - JOUR AB - Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch transactivate genes by interacting with the transcription factor RBP-Jkappa. The viral protein EBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor. Until now, nothing has been known about the physiological role of Notch signaling in B cells. Here we investigated whether activated Notch can induce the same phenotypic changes as EBNA2 in Burkitt's lymphoma cells. An estrogen receptor fusion protein of the intracellular part of mouse Notch 1 (mNotch1-IC), mimicking in the presence of estrogen a constitutively active Notch receptor, was stably transfected into the Burkitt's lymphoma cell lines BL41-P3HR1 and HH514. Northern blot analysis revealed that the LMP2A gene is induced by Notch-IC in the presence of estrogen, whereas increased expression of LMP1 could be detected only if cycloheximide was simultaneously added. Concerning the cellular genes regulated by EBNA2, Notch-IC was able to upregulate CD21 but not CD23 expression. Immunoglobulin mu (Igmu) expression, which is downregulated by EBNA2, was also negatively regulated by Notch-IC. Similarly to EBNA2, Notch-IC was able to repress c-myc expression, which is under the control of the immunoglobulin heavy-chain locus in Burkitt's lymphoma cells with a t(8;14) translocation. The data show that Notch-IC is able to participate in gene regulation in B cells. AU - Strobl, L.J. AU - Höfelmayr, H. AU - Marschall, G. AU - Brielmeier, M. AU - Bornkamm, G.W. AU - Zimber-Strobl, U. C1 - 10022 C2 - 22867 SP - 1727-1735 TI - Activated Notch1 modulates gene expression in B cells similary to Epstein-Barr viral nuclear antigen 2. JO - J. Virol. VL - 74 IS - 4 PY - 2000 SN - 0022-538X ER - TY - JOUR AB - Astrocytes are target cells for human immunodeficiency virus type 1 (HIV-1) in the central nervous system with attenuated virus replication in vivo and in vitro. In infected astrocytes, viral gene expression is restricted mainly to nonstructural (early) viral components like Nef, suggesting inhibition of Rev-dependent posttranscriptional processes in these cells. Because of the heterogeneity of astrocytic cells, the objective of this study was to determine whether restriction of HIV-1 Rev-associated activities is a common property of human astrocytes. To this end, we compared the trans activation capacity and intracellular distribution of Rev in four astrocytoma cell lines previously shown to be infectible by HIV-1 and in primary human fetal astrocytes from different sources with Rev-permissive nonglial control cell lines. In all astrocytic cell cultures, the Rev response was reduced to about 10% of that of Rev-permissive control cells. Rev was apparent both in cytoplasmic and in nuclear compartments of living astrocytes, in contrast to the typical nuclear and/or nucleolar localization of Rev in permissive control cells. Nuclear accumulation of Rev in astrocytes was restored by blocking export of Rev. The trans activation capacity and nuclear localization of Tat were not affected in astrocytes. These results demonstrate that inhibition of Rev-dependent posttranscriptional regulation of HIV-1 is a hallmark of human astrocytes and may contribute to suppression of HIV-1 production in these HIV-1 reservoirs. Astrocytes constitute the first example of a human cell type showing an impaired Rev response, indicating that posttranscriptional control of HIV-1 gene expression can be modulated in a cell-dependent manner. AU - Ludwig, E. AU - Silberstein, F.C. AU - van Empel, J. AU - Erfle, V. AU - Neumann, M. AU - Brack-Werner, R. C1 - 24076 C2 - 31437 SP - 8279-8289 TI - Diminished rev-mediated stimulation of human immunodeficiency virus type 1 protein synthesis is a hallmark of human astrocytes. JO - J. Virol. VL - 73 IS - 10 PB - Amer. Soc. Microbiology PY - 1999 SN - 0022-538X ER - TY - JOUR AU - Nevels, M.* AU - Täuber, B.* AU - Kremmer, E. AU - Spruss, T.* AU - Wolf, H.* AU - Dobner, T.* C1 - 20869 C2 - 18923 SP - 1591-1600 TI - Transforming potential of the adenovirus type 5 E4orf3 protein. JO - J. Virol. VL - 73 PY - 1999 SN - 0022-538X ER - TY - JOUR AB - BZLF1 is a member of the extended AP-1 family of transcription factors which binds to specific BZLF1 sequence motifs within early Epstein-Barr virus (EBV) promoters and to closely related AP-1 motifs. BZLF1's activity is regulated at the transcriptional level as well as through protein interactions and posttranslational modifications. Phorbol esters or immunoglobulin cross-linking both reactivate EBV from latently infected B cells via transactivation of BZLF1. We report here that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is capable of inducing BZLF1's activity even further. The induction occurs at the posttranscriptional level and depends on a single serine residue located in the DNA binding domain of BZLF1. This serine residue (S186) is phosphorylated by protein kinase C in vitro and in vivo after stimulation with TPA. Phosphorylation of S186 per se interferes with the DNA binding affinity of BZLF1 in vitro but is mandatory for TPA-induced increase in DNA binding of BZLF1, as shown in gel retardation assays and reconstruction experiments with cellular extracts. In transcriptional reporter assays, S186 is essential for the activation of BZLF1 by TPA. Presumably, a yet-to-be-identified cellular factor restores the DNA binding affinity and enhances the transcriptional activity of S186-phosphorylated BZLF1, which is required to induce the lytic phase of EBV's life cycle. AU - Baumann, M. AU - Mischak, H. AU - Dammeier, S. AU - Kolch, W. AU - Gires, O. AU - Pich, D. AU - Zeidler, R. AU - Delecluse, H.-J. AU - Hammerschmidt, W. C1 - 27632 C2 - 32776 SP - 8105-8114 TI - Activation of the Epstein-Barr virus transcription factor BZLF1 by 12-O-tetradecanoylphorbol-13-acetate-induced phosphorylation. JO - J. Virol. VL - 72 IS - 10 PY - 1998 SN - 0022-538X ER - TY - JOUR AB - We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid. These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus. One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame. When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus. Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus. The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes. These findings identify 41% of EBV DNA which is sufficient to immortalize primary human B lymphocytes and provide an assay to distinguish virus contributions to initiation or maintenance of cell proliferation or both. They also identify EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation. AU - Kempkes, B. AU - Pich, D. AU - Zeidler, R. AU - Sugden, B.* AU - Hammerschmidt, W. C1 - 27612 C2 - 32766 SP - 231-238 TI - Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA. JO - J. Virol. VL - 69 IS - 1 PY - 1995 SN - 0022-538X ER - TY - JOUR AB - EBNA2 is one of the few genes of Epstein-Barr virus which are necessary for immortalization of human primary B lymphocytes. The EBNA2 protein acts as a transcriptional activator of several viral and cellular genes. For the TP1 promoter, we have shown previously that an EBNA2-responsive element (EBNA2RE) between -258 and -177 relative to the TP1 RNA start site is necessary and sufficient for EBNA2-mediated transactivation and that it binds EBNA2 through a cellular factor. To define the critical cis elements within this region, we cloned EBNA2RE mutants in front of the TP1 minimal promoter fused to the reporter gene for luciferase. Transactivation by EBNA2 was tested by transfection of these mutants in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR-1. The analysis revealed that two identical 11-bp motifs and the region 3' of the second 11- bp motif are essential for transactivation by EBNA2. Methylation interference experiments indicated that the same cellular factor in the absence of EBNA2 binds either one (complex I) or both (complex III) 11-bp motifs with different affinities, giving rise to two different specific protein-DNA complexes within the left-hand 54 bp of EBNA2RE. A third specific complex was shown previously to be present only in EBNA2-expressing cells and to contain EBNA2. Analysis of this EBNA2-containing complex revealed the same protection pattern as for complex III, indicating that EBNA2 interacts with DNA through binding of the cellular protein to the 11-bp motifs. Mobility shift assays with the different mutants demonstrated that one 11-bp motif is sufficient for binding the cellular factor, whereas for binding of EBNA2 as well as for efficient transactivation by EBNA2, both 11-bp motifs are required. AU - Meitinger, C. AU - Strobl, L.J. AU - Marschall, G. AU - Bornkamm, G.W. AU - Zimber-Strobl, U. C1 - 40048 C2 - 0 SP - 7497-7506 TI - Crucial sequences within the Epstein-Barr virus TP1 promoter for EBNA2- mediated transactivation and interaction of EBNA2 with its responsive element. JO - J. Virol. VL - 68 IS - 11 PY - 1994 SN - 0022-538X ER - TY - JOUR AU - Bornkamm, G.W. AU - Delecluse, H.-J. AU - Hammerschmidt, W. AU - Bullerdiek, J. C1 - 20125 C2 - 13301 SP - 1292-1299 TI - Episomal and Integrated Copies of Epstein-Barr Virus Coexist in Burkitt Lymphoma Cell Lines. JO - J. Virol. VL - 67 PY - 1993 SN - 0022-538X ER - TY - JOUR AB - The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated. AU - Delecluse, H.J. AU - Bartnizke, S.* AU - Hammerschmidt, W. AU - Bullerdiek, J.O.* AU - Bornkamm, G.W. C1 - 40306 C2 - 40075 SP - 1292-1299 TI - Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines. JO - J. Virol. VL - 67 IS - 3 PY - 1993 SN - 0022-538X ER - TY - JOUR AB - Six cell lines derived from Marek's disease lymphomas of chickens and turkeys were investigated for the status of Marek's disease virus (MDV) DNA. In the transformed T- and B-cell lines, viral DNA could be detected by conventional Southern blot hybridization, by Gardella gel electrophoresis, and by in situ hybridization of metaphase and interphase chromosomes. Integration of viral DNA into the host cell chromosome was observed in all cell lines. Two to 12 integration sites of viral DNA could be detected in metaphase chromosome spreads. The integration sites were characteristic for the individual cell lines and were preferentially located at the telomers of large- and mid-sized chromosomes or on minichromosomes. In four of six cell lines, a minor population of latently infected cells supported the lytic cycle of MDV, giving rise to linear virion DNAs. In one of these cell lines, a third species of MDV DNA could be detected with properties reminiscent of covalently closed circular DNA. The finding that MDV integrates regularly into the genomes of latently infected cells is crucial to understanding the molecular biology of herpesvirus-induced tumors in the natural host. AU - Delecluse, H.J. AU - Hammerschmidt, W. C1 - 40326 C2 - 38006 SP - 82-92 TI - Status of Marek's disease virus in established lymphoma cell lines: Herpesvirus integration is common. JO - J. Virol. VL - 67 IS - 1 PY - 1993 SN - 0022-538X ER - TY - JOUR AU - Schepers, A. AU - Pich, D. AU - Mankertz, J. AU - Hammerschmidt, W. C1 - 20452 C2 - 13658 SP - 4237-4245 TI - Cis-acting Elements in the Lytic Origin of DNA Replication of Epstein-Barr Virus. JO - J. Virol. VL - 67 PY - 1993 SN - 0022-538X ER - TY - JOUR AB - oriLyt, the cis-acting element of Epstein-Barr virus, mediates viral DNA replication in the lytic phase of the virus's life cycle. Oligonucleotide-directed in vitro mutagenesis of oriLyt plasmids allowed the identification of two noncontiguous components within the complex structure of oriLyt. Both components were indispensable for DNA replication of this origin. The upstream component colocalized with the promoter of the viral BHLF1-encoding gene, and mutants affecting DNA replication affected RNA transcription, too. The second component crucial for oriLyt function was determined to be 40 bp long and positioned approximately 530 bp downstream. It was dispensable for transcriptional transactivation but it was absolutely required for replication. Thus, the overall design of oriLyt has striking similarity to multipartite regulatory elements of transcription, consisting of proximal promoters and distal enhancers, but special elements are exclusively dedicated to DNA replication. AU - Schepers, A. AU - Pich, D. AU - Mankertz, J. AU - Hammerschmidt, W. C1 - 40356 C2 - 40042 SP - 4237-4245 TI - cis-acting elements in the lytic origin of DNA replication of Epstein-Barr virus. JO - J. Virol. VL - 67 IS - 7 PY - 1993 SN - 0022-538X ER - TY - JOUR AB - T-cell subsets were studied by fluorescence-activated cell sorter analysis in 57 feline immunodeficiency virus (FIV)-seropositive cats with naturally acquired FIV infection to see whether CD4+-CD8+ alterations were comparable to those observed in human immunodeficiency virus-infected patients. CD4+ values were decreased and CD8+ values were increased. The CD4+/CD8+ ratio was reduced to 1.6, compared with 3.3 in 33 FIV-seronegative control cats. Variance analysis of data showed a significant influence of FIV seroposilivity, sex, and spaying of female cats on CD4+ values. CD8+ values were significantly influenced by FIV seropositivity, age, and breed. These findings indicate a similarity between FIV and human immunodeficiency virus infections, as far as alterations of T-cell subsets are concerned. AU - Hoffmann-Fezer, G. AU - Thum, J. AU - Ackley, C.D.* AU - Herbold, M. AU - Mysliwietz, J. AU - Thefeld, S. AU - Hartmann, K.I.* AU - Kraft, W.* C1 - 40577 C2 - 38771 SP - 1484-1488 TI - Decline in CD4+ cell numbers in cats with naturally acquired feline immunodeficiency virus infection. JO - J. Virol. VL - 66 IS - 3 PY - 1992 SN - 0022-538X ER - TY - JOUR AU - Zimber-Strobl, U. AU - Suentzenich, K.-O. AU - Laux, G. AU - Eick, D. AU - Cordier, M. AU - Calender, A. AU - Billaud, M. AU - Lenoir, G.M. AU - Bornkamm, G.W. C1 - 19602 C2 - 12711 SP - 415-423 TI - Epstein-Barr Virus Nuclear Antigen 2 Activates Transcription of the Terminal Protein Gene. JO - J. Virol. VL - 65 PY - 1991 SN - 0022-538X ER - TY - JOUR AU - Cordier, M. AU - Calender, A. AU - Billaud, M. AU - Zimber-Srobl, U. AU - Rousselet, G. AU - Pavlish, O. AU - Banchereau, J. AU - Tursz, T. AU - Bornkamm, G.W. AU - Lenoir, G.M. C1 - 19269 C2 - 12342 SP - 1002-1013 TI - Stable Transfection of Epstein-Barr Virus (EBV) Nuclear Antigen 2 in Lymphoma Cells Containing the EBV P3HR1 Genome Induces Expression of B-Cell Activation Molecules CD21 and CD23. JO - J. Virol. VL - 64 PY - 1990 SN - 0022-538X ER - TY - JOUR AU - Frech, B. AU - Zimber-Strobl, U. AU - Suentzenich, K.-O. AU - Pavlish, O. AU - Lenoir, G.M. AU - Bornkamm, G.W. AU - Müller-Lantzsch, N. C1 - 19268 C2 - 12341 SP - 2759-2767 TI - Identification of Epstein-Barr Virus Terminal Protein 1 (TP1) in Extracts of Four Lymphoid Cell Lines, Expression in Insect Cells, and Detection of Antibodies in Human Sera. JO - J. Virol. VL - 64 PY - 1990 SN - 0022-538X ER - TY - JOUR AU - Rowe, D.T. AU - Hall, L. AU - Joab, I. AU - Laux, G. C1 - 18836 C2 - 11956 SP - 2866-2875 TI - Identification of the Epstein-Barr Virus Terminal Protein Gene Products in Latently Infected Lymphocytes. JO - J. Virol. VL - 64 PY - 1990 SN - 0022-538X ER - TY - JOUR AU - Salmons, B. AU - Erfle, V. AU - Brem, G. AU - Günzburg, W.H. C1 - 18574 C2 - 11702 TI - naf, a Trans-regulating Negative Acting Factor Encoded within the Mouse Mammary Tumour Virus 0RF Region. JO - J. Virol. PY - 1990 SN - 0022-538X ER - TY - JOUR AB - The mouse mammary tumor virus (MMTY) long terminal repeat (LTR) open reading frame (ORF) encodes a nagative acting factor (naf). In our test system, naf mediates its effect in trans on another MMTV provirus in which the 5′ LTM has been replaced by that of Rous sarcoma virus. naf effects are evidenced at the evidenced at the level of transcriptional initiation rather than as reduced mRNA stability. The introduction of a premature termination codon into the MMTV LTR-encoded ORF abolishes the transcriptional down regulation localizing naf within the ORF. In addition, sequences in the gag/pol genes between + 320 and + 646 and between + 3626 and + 4590 relative to the site of transcription initiation are also involved in the MMTV- mediated transcriptional down regulation. AU - Salmons, B.* AU - Erfle, V.F. AU - Brem, G.* AU - Günzburg, W.H. C1 - 33996 C2 - 35438 SP - 6355-6359 TI - Naf, a trans-regulating negative-acting factor encoded within the mouse mammary tumor virus open reading frame region. JO - J. Virol. VL - 64 IS - 12 PY - 1990 SN - 0022-538X ER - TY - JOUR AB - The rate of endolysin synthesis in Salmonella typhimurium cells infected by bacteriophage P22 or L was taken as a measure for the activity of 23 gene product (the positive regulator for the 'late' genes of P22 and L). Endolysin is coded for by gene 19. The amber mutations in gene 23 of P22 and L, used in this study, reduced the rate of endolysin synthesis by a factor of ca. 90 or P22 and of ca. 20 for L. In mixed infections with 19- and 23- mutants the 23 gene products of P22 and L act as positive regulators for the respective gene 19 in cis and in trans. Cross-specificity of the 23 gene products, i.e. turning on expression of gene 19 on a chromosome of the other species, could not be demonstrated. AU - Bode, W. C1 - 41084 C2 - 35757 SP - 1-7 TI - Regulation of late functions in Salmonella bateriophages P22 and L studied by assaying endolysin synthesis. JO - J. Virol. VL - 32 IS - 1 PY - 1979 SN - 0022-538X ER -