TY - JOUR AB - Courses of SARS-CoV-2 infections are highly variable, ranging from asymptomatic to lethal COVID-19. Though research has shown that host genetic factors contribute to this variability, cohort-based joint analyses of variants from the entire allelic spectrum in individuals with confirmed SARS-CoV-2 infections are still lacking. Here, we present the results of whole genome sequencing in 1,220 mainly vaccine-naïve individuals with confirmed SARS-CoV-2 infection, including 827 hospitalized COVID-19 cases. We observed the presence of autosomal-recessive or likely compound heterozygous monogenic disorders in six individuals, all of which were hospitalized and significantly younger than the rest of the cohort. We did not observe any suggestive causal variants in or around the established risk gene TLR7. Burden testing in the largest population subgroup (i.e., Europeans) suggested nominal enrichments of rare variants in coding and non-coding regions of interferon immune response genes in the overall analysis and male subgroup. Case-control analyses of more common variants confirmed associations with previously reported risk loci, with the key locus at 3p21 reaching genome-wide significance. Polygenic scores accurately captured risk in an age-dependent manner. By enabling joint analyses of different types of variation across the entire frequency spectrum, this data will continue to contribute to the elucidation of COVID-19 etiology. AU - Schmidt, A.* AU - Casadei, N.* AU - Brand, F.* AU - Demidov, G.* AU - Vojgani, E.* AU - Abolhassani, A.* AU - Aldisi, R.* AU - Butler-Laporte, G.* AU - Alawathurage, T.M.* AU - Augustin, M.* AU - Bals, R.* AU - Bellinghausen, C.* AU - Berger, M.M.* AU - Bitzer, M.* AU - Bode, C.* AU - Boos, J.* AU - Brenner, T.* AU - Cornely, O.A.* AU - Eggermann, T.* AU - Erber, J.* AU - Feldt, T.* AU - Fuchsberger, C.* AU - Gagneur, J. AU - Göpel, S.* AU - Haack, T.* AU - Häberle, H.* AU - Hanses, F.* AU - Heggemann, J.* AU - Hehr, U.* AU - Hellmuth, J.C.* AU - Herr, C.* AU - Hinney, A.* AU - Hoffmann, P.* AU - Illig, T.* AU - Jensen, B.O.* AU - Keitel, V.* AU - Kim-Hellmuth, S. AU - Koehler, P.* AU - Kurth, I.* AU - Lanz, A.L.* AU - Latz, E.* AU - Lehmann, C.* AU - Luedde, T.* AU - Maj, C.* AU - Mian, M.* AU - Miller, A.* AU - Muenchhoff, M.* AU - Pink, I.* AU - Protzer, U. AU - Rohn, H.* AU - Rybniker, J.* AU - Scaggiante, F.* AU - Schaffeldt, A.* AU - Scherer, C.* AU - Schieck, M.* AU - Schmidt, S.V.* AU - Schommers, P.* AU - Spinner, C.D.* AU - Vehreschild, M.J.G.T.* AU - Velavan, T.P.* AU - Volland, S.* AU - Wilfling, S.* AU - Winter, C.* AU - Richards, J.B.* AU - Heimbach, A.* AU - Becker, K.* AU - Ossowski, S.* AU - Schultze, J.L.* AU - Nürnberg, P.* AU - Nöthen, M.M.* AU - Motameny, S.* AU - Nothnagel, M.* AU - Riess, O.* AU - Schulte, E.C. AU - Ludwig, K.U.* C1 - 72891 C2 - 56802 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Systematic assessment of COVID-19 host genetics using whole genome sequencing data. JO - PLoS Pathog. VL - 20 IS - 12 PB - Public Library Science PY - 2024 SN - 1553-7366 ER - TY - JOUR AB - Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific cell types and brain regions. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite appearing normal. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated reduced synthesis of ribosomal and mitochondrial components, 2) glutamatergic neurons showed increased expression of cytoskeletal genes, and 3) GABAergic neurons revealed reduced expression of circadian rhythm genes. These data demonstrate that early translatome responses to neurodegeneration emerge prior to conventional markers of disease and are cell type-specific. Therapeutic strategies may need to target multiple pathways in specific populations of cells, early in disease. AU - Kaczmarczyk, L.* AU - Schleif, M.* AU - Dittrich, L.* AU - Williams, R.H. AU - Koderman, M.* AU - Bansal, V.* AU - Rajput, A.* AU - Schulte, T.L.* AU - Jonson, M.* AU - Krost, C.* AU - Testaquadra, F.J.* AU - Bonn, S.* AU - Jackson, W.S.* C1 - 66016 C2 - 53056 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Distinct translatome changes in specific neural populations precede electroencephalographic changes in prion-infected mice. JO - PLoS Pathog. VL - 18 IS - 8 PB - Public Library Science PY - 2022 SN - 1553-7366 ER - TY - JOUR AB - Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) "histone code" for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx. AU - Kim, E.S.* AU - Zhou, J.* AU - Zhang, H.* AU - Marchetti, A.* AU - van de Klundert, M. AU - Cai, D.* AU - Yu, X.* AU - Mitra, B.* AU - Liu, Y.* AU - Wang, M.* AU - Protzer, U. AU - Guo, H.* C1 - 65415 C2 - 52457 TI - Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA. JO - PLoS Pathog. VL - 18 IS - 6 PY - 2022 SN - 1553-7366 ER - TY - JOUR AB - Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies. AU - Akidil, E. AU - Albanese, M. AU - Buschle, A. AU - Ruhle, A.* AU - Pich, D. AU - Keppler, O.T.* AU - Hammerschmidt, W. C1 - 61850 C2 - 50194 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection. JO - PLoS Pathog. VL - 17 IS - 4 PB - Public Library Science PY - 2021 SN - 1553-7366 ER - TY - JOUR AB - Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients. AU - Winheim, E.* AU - Rinke, L.* AU - Lutz, K.* AU - Reischer, A.* AU - Leutbecher, A.* AU - Wolfram, L.* AU - Rausch, L.* AU - Kranich, J.* AU - Wratil, P.R.* AU - Huber, J.E.* AU - Baumjohann, D.* AU - Rothenfußer, S. AU - Schubert, B. AU - Hilgendorff, A. AU - Hellmuth, J.C.* AU - Scherer, C.* AU - Muenchhoff, M.* AU - von Bergwelt-Baildon, M.* AU - Stark, K.* AU - Straub, T.* AU - Brocker, T.* AU - Keppler, O.T.* AU - Subklewe, M.* AU - Krug, A.B.* C1 - 63298 C2 - 51459 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Impaired function and delayed regeneration of dendritic cells in COVID-19. JO - PLoS Pathog. VL - 17 IS - 10 PB - Public Library Science PY - 2021 SN - 1553-7366 ER - TY - JOUR AB - Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA. To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo. Using co-infection experiments and recombinant viruses, we also show that KSHV'S ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.Author summary During herpesvirus latency, viral genomes persist as partially repressed nuclear episomes which do not express genes required for progeny production. Latently infected cells not only form a reservoir of lifelong persistence but also represent the driving force in cancers associated with tumorigenic herpesviruses such as KSHV. Hence, it is fundamentally important to understand the mechanisms controlling latency. We have shown previously that latent KSHV episomes rapidly acquire H3K27-me3, a histone mark associated with polycomb repressive complexes (PRC). PRCs play a pivotal role in the control of developmental genes but are also involved in the pathogenesis of several tumors. We here investigated whether PRC-repression represents a general feature of herpesvirus latency. By performing side-by-side analyses of KSHV and the related MHV-68 we show that the latter indeed has a fundamentally lower propensity to acquire H3K27-me3, and that KSHV'S ability to rapidly attract this mark is most likely the result of a specific sequence composition that promotes recruitment of non-canonical PRC1 (a complex which is important for the regulation of cellular CpG islands). Our results have widespread implications for nuclear DNA viruses and suggest that some viruses have specifically evolved to exploit common host responses to epigenetically naive DNA. AU - Günther, T.* AU - Fröhlich, J.* AU - Herrde, C.* AU - Ohno, S. AU - Burkhardt, L.* AU - Adler, H. AU - Grundhoff, A.* C1 - 57239 C2 - 47628 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment. JO - PLoS Pathog. VL - 15 IS - 10 PB - Public Library Science PY - 2019 SN - 1553-7366 ER - TY - JOUR AB - Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression. AU - Koch, S.* AU - Damas, M.* AU - Freise, A.* AU - Hage, E.* AU - Dhingra, A.* AU - Rückert, J.* AU - Gallo, A.* AU - Kremmer, E. AU - Tegge, W.* AU - Brönstrup, M.* AU - Brune, W.* AU - Schulz, T.F.* C1 - 55993 C2 - 46748 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Kaposi's sarcoma-associated herpesvirus vIRF2 protein utilizes an IFN-dependent pathway to regulate viral early gene expression. JO - PLoS Pathog. VL - 15 IS - 5 PB - Public Library Science PY - 2019 SN - 1553-7366 ER - TY - JOUR AU - Hufsky, F.* AU - Ibrahim, B.* AU - Beer, M.* AU - Deng, L. AU - Mercier, P.L.* AU - McMahon, D.P.* AU - Palmarini, M.* AU - Thiel, V.* AU - März, M.* C1 - 53211 C2 - 44509 CY - San Francisco TI - Virologists-Heroes need weapons. JO - PLoS Pathog. VL - 14 IS - 2 PB - Public Library Science PY - 2018 SN - 1553-7366 ER - TY - JOUR AB - Human herpesvirus 6 (HHV-6) is prevalent in healthy persons, causes disease in immunosuppressed carriers, and may be involved in autoimmune disease. Cytotoxic CD8 T cells are probably important for effective control of infection. However, the HHV-6-specific CD8 T cell repertoire is largely uncharacterized. Therefore, we undertook a virus-wide analysis of CD8 T cell responses to HHV-6. We used a simple anchor motif-based algorithm (SAMBA) to identify 299 epitope candidates potentially presented by the HLA class I molecule B*08:01. Candidates were found in 77 of 98 unique HHV-6B proteins. From peptide-expanded T cell lines, we obtained CD8 T cell clones against 20 candidates. We tested whether T cell clones recognized HHV-6-infected cells. This was the case for 16 epitopes derived from 12 proteins from all phases of the viral replication cycle. Epitopes were enriched in certain amino acids flanking the peptide. Ex vivo analysis of eight healthy donors with HLA-peptide multimers showed that the strongest responses were directed against an epitope from IE-2, with a median frequency of 0.09% of CD8 T cells. Reconstitution of T cells specific for this and other HHV-6 epitopes was also observed after allogeneic hematopoietic stem cell transplantation. We conclude that HHV-6 induces CD8 T cell responses against multiple antigens of diverse functional classes. Most antigens against which CD8 T cells can be raised are presented by infected cells. Ex vivo multimer staining can directly identify HHV-6-specific T cells. These results will advance development of immune monitoring, adoptive T cell therapy, and vaccines. AU - Martin, L.K. AU - Hollaus, A. AU - Stahuber, A. AU - Hübener, C. AU - Fraccaroli, A. AU - Tischer, J. AU - Schub, A. AU - Moosmann, A. C1 - 53453 C2 - 44594 CY - 233 Spring St, New York, Ny 10013 Usa TI - Cross-sectional analysis of CD8 T cell immunity to human herpesvirus 6B. JO - PLoS Pathog. VL - 14 IS - 4 PB - Springer PY - 2018 SN - 1553-7366 ER - TY - JOUR AB - Innate immune recognition is classically mediated by the interaction of host pattern-recognition receptors and pathogen-associated molecular patterns; this triggers a series of downstream signaling events that facilitate killing and elimination of invading pathogens. In this report, we provide the first evidence that peroxidasin (PXDN; also known as vascular peroxidase-1) directly binds to gram-negative bacteria and mediates bactericidal activity, thus, contributing to lung host defense. PXDN contains five leucine-rich repeats and four immunoglobulin domains, which allows for its interaction with lipopolysaccharide, a membrane component of gram-negative bacteria. Bactericidal activity of PXDN is mediated via its capacity to generate hypohalous acids. Deficiency of PXDN results in a failure to eradicate Pseudomonas aeruginosa and increased mortality in a murine model of Pseudomonas lung infection. These observations indicate that PXDN mediates previously unrecognized host defense functions against gram-negative bacterial pathogens. AU - Shi, R.* AU - Cao, Z.* AU - Li, H.* AU - Graw, J. AU - Zhang, G.* AU - Thannickal, V.J.* AU - Cheng, G.* C1 - 53549 C2 - 44891 CY - 1200 New York Ave, Nw, Washington, Dc 20005 Usa TI - Peroxidasin contributes to lung host defense by direct binding and killing of gram-negative bacteria. JO - PLoS Pathog. VL - 14 IS - 5 PB - Amer Assoc Advancement Science PY - 2018 SN - 1553-7366 ER - TY - JOUR AB - The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection. AU - van Zyl, D.G.* AU - Tsai, M.* AU - Shumilov, A.* AU - Schneidt, V.* AU - Poirey, R.* AU - Schlehe, B.* AU - Fluhr, H.* AU - Mautner, J. AU - Delecluse, H.-J.* C1 - 55208 C2 - 46091 CY - 1160 Battery Street, Ste 100, San Francisco, Ca 94111 Usa TI - Immunogenic particles with a broad antigenic spectrum stimulate cytolytic T cells and offer increased protection against EBV infection ex vivo and in mice. JO - PLoS Pathog. VL - 14 IS - 12 PB - Public Library Science PY - 2018 SN - 1553-7366 ER - TY - JOUR AB - Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2. AU - Glaser, L.V. AU - Rieger, S. AU - Thumann, S. AU - Beer, S. AU - Kuklik-Roos, C. AU - Martin, D.E.* AU - Maier, K.C.* AU - Harth-Hertle, M.L. AU - Grüning, B.* AU - Backofen, R.* AU - Krebs, S.* AU - Blum, H.* AU - Zimmer, R.* AU - Erhard, F.* AU - Kempkes, B. C1 - 52025 C2 - 43670 CY - San Francisco SP - e1006664 TI - EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells. JO - PLoS Pathog. VL - 13 IS - 10 PB - Public Library Science PY - 2017 SN - 1553-7366 ER - TY - JOUR AB - Fusarium fujikuroi causes bakanae ("foolish seedling") disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles. AU - Niehaus, E.M.* AU - Kim, H.K.* AU - Münsterkötter, M. AU - Janevska, S.* AU - Arndt, B.* AU - Kalinina, S.A.* AU - Houterman, P.M.* AU - Ahn, I.P.* AU - Alberti, I.* AU - Tonti, S.* AU - Kim, D.W.* AU - Sieber, C.M.K.* AU - Humpf, H.U.* AU - Yun, S.H.* AU - Güldener, U. AU - Tudzynski, B.* C1 - 52216 C2 - 43857 CY - San Francisco TI - Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles. JO - PLoS Pathog. VL - 13 IS - 10 PB - Public Library Science PY - 2017 SN - 1553-7366 ER - TY - JOUR AB - Human cytomegalovirus (CMV) infection is a substantial cause of morbidity and mortality in immunocompromised hosts and globally is one of the most important congenital infections. The nucleoside analogue ganciclovir (GCV), which requires initial phosphorylation by the viral UL97 kinase, is the mainstay for treatment. To date, CMV decay kinetics during GCV therapy have not been extensively investigated and its clinical implications not fully appreciated. We measured CMV DNA levels in the blood of 92 solid organ transplant recipients with CMV disease over the initial 21 days of ganciclovir therapy and identified four distinct decay patterns, including a new pattern exhibiting a transient viral rebound (Hump) following initial decline. Since current viral dynamics models were unable to account for this Hump profile, we developed a novel multi-level model, which includes the intracellular role of UL97 in the continued activation of ganciclovir, that successfully described all the decline patterns observed. Fitting the data allowed us to estimate ganciclovir effectiveness in vivo (mean 92%), infected cell half-life (mean 0.7 days), and other viral dynamics parameters that determine which of the four kinetic patterns will ensue. An important clinical implication of our results is that the virological efficacy of GCV operates over a broad dose range. The model also raises the possibility that GCV can drive replication to a new lower steady state but ultimately cannot fully eradicate it. This model is likely to be generalizable to other anti-CMV nucleoside analogs that require activation by viral enzymes such as UL97 or its homologues. AU - Rose, J.* AU - Emery, V.C.* AU - Kumar, D.* AU - Asberg, A.* AU - Hartmann, A.* AU - Jardine, A.G.* AU - Bignamini, A.A.* AU - Humar, A.* AU - Neumann, A.U. C1 - 51377 C2 - 43043 CY - San Francisco TI - Novel decay dynamics revealed for virus-mediated drug activation in cytomegalovirus infection. JO - PLoS Pathog. VL - 13 IS - 4 PB - Public Library Science PY - 2017 SN - 1553-7366 ER - TY - JOUR AB - The Epstein-Barr virus (EBV) infects and transforms B-lymphocytes with high efficiency. This process requires expression of the viral latent proteins and of the 3 miR-BHRF1 microRNAs. Here we show that B-cells infected by a virus that lacks these non-coding RNAs (Δ123) grew more slowly between day 5 and day 20, relative to wild type controls. This effect could be ascribed to a reduced S phase entry combined with a moderately increased apoptosis rate. Whilst the first phenotypic trait was consistent with an enhanced PTEN expression in B-cells infected with Δ123, the second could be explained by very low BHRF1 protein and RNA levels in the same cells. Indeed, B-cells infected either by a recombinant virus that lacks the BHRF1 protein, a viral bcl-2 homolog, or by Δ123 underwent a similar degree of apoptosis, whereas knockouts of both BHRF1 microRNAs and protein proved transformation-incompetent. We find that that the miR-BHRF1-3 seed regions, and to a lesser extent those of miR-BHRF1-2 mediate these stimulatory effects. After this critical period, B-cells infected with the Δ123 mutant recovered a normal growth rate and became more resistant to provoked apoptosis. This resulted from an enhanced BHRF1 protein expression relative to cells infected with wild type viruses and correlated with decreased p27 expression, two pro-oncogenic events. The upregulation of BHRF1 can be explained by the observation that large BHRF1 mRNAs are the source of BHRF1 protein but are destroyed following BHRF1 microRNA processing, in particular of miR-BHRF1-2. The BHRF1 microRNAs are unlikely to directly target p27 but their absence may facilitate the selection of B-cells that express low levels of this protein. Thus, the BHRF1 microRNAs allowed a time-restricted expression of the BHRF1 protein to innocuously expand the virus B-cell reservoir during the first weeks post-infection without increasing long-term immune pressure. AU - Bernhardt, K.* AU - Haar, J.* AU - Tsai, M.H.* AU - Poirey, R.* AU - Feederle, R. AU - Delecluse, H.-J.* C1 - 50320 C2 - 42097 TI - A viral microRNA cluster regulates the expression of PTEN, p27 and of a bcl-2 homolog. JO - PLoS Pathog. VL - 12 IS - 1 PY - 2016 SN - 1553-7366 ER - TY - JOUR AB - It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression-particularly in relation to histone modifications and cell factors involved-the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells. AU - Kalchschmidt, J.S.* AU - Gillman, A.C.* AU - Paschos, K.* AU - Bazot, Q.* AU - Kempkes, B. AU - Allday, M.J.* C1 - 47669 C2 - 39716 CY - San Francisco TI - EBNA3C directs recruitment of RBPJ (CBF1) to chromatin during the process of gene repression in EBV infected B cells. JO - PLoS Pathog. VL - 12 IS - 1 PB - Public Library Science PY - 2016 SN - 1553-7366 ER - TY - JOUR AB - Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer’s patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host’s intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies. AU - Nuss, A.M.* AU - Schuster, F.* AU - Roselius, L.* AU - Klein, J.* AU - Bücker, R.* AU - Herbst, K.* AU - Heroven, A.K.* AU - Pisano, F.* AU - Wittmann, C.* AU - Münch, R.* AU - Müller, J. AU - Jahn, D.* AU - Dersch, P.* C1 - 50284 C2 - 42253 CY - San Francisco TI - A precise temperature-responsive bistable switch controlling yersinia virulence. JO - PLoS Pathog. VL - 12 IS - 12 PB - Public Library Science PY - 2016 SN - 1553-7366 ER - TY - JOUR AB - An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues. AU - Sattler, C. AU - Steer, B. AU - Adler, H. C1 - 48183 C2 - 39947 CY - San Francisco TI - Multiple lytic origins of replication are required for optimal gammaherpesvirus fitness in vitro and in vivo. JO - PLoS Pathog. VL - 12 IS - 3 PB - Public Library Science PY - 2016 SN - 1553-7366 ER - TY - JOUR AB - Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing. AU - Esser-von Bieren, J. AU - Volpe, B.* AU - Sutherland, D.B.* AU - Bürgi, J.* AU - Verbeek, J.S.* AU - Marsland, B.J.* AU - Urban, J.F.* AU - Harris, N.L.* C1 - 43975 C2 - 36703 CY - San Francisco TI - Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair. JO - PLoS Pathog. VL - 11 IS - 3 PB - Public Library Science PY - 2015 SN - 1553-7366 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics. AU - Friberg, A. AU - Thumann, S. AU - Hennig, J. AU - Zou, P. AU - Nößner, E. AU - Ling, P.D.* AU - Sattler, M. AU - Kempkes, B. C1 - 45053 C2 - 37190 CY - San Francisco TI - The EBNA-2 N-terminal transactivation domain folds into a dimeric structure required for target gene activation. JO - PLoS Pathog. VL - 11 IS - 5 PB - Public Library Science PY - 2015 SN - 1553-7366 ER - TY - JOUR AB - Kaposi's sarcoma (KS), caused by Kaposi's sarcoma herpesvirus (KSHV), is a highly vascularised tumour of endothelial origin. KSHV infected endothelial cells show increased invasiveness and angiogenesis. Here, we report that the KSHV K15 protein, which we showed previously to contribute to KSHV-induced angiogenesis, is also involved in KSHV-mediated invasiveness in a PLCγ1-dependent manner. We identified βPIX, GIT1 and cdc42, downstream effectors of PLCγ1 in cell migration, as K15 interacting partners and as contributors to KSHV-triggered invasiveness. We mapped the interaction between PLCγ1, PLCγ2 and their individual domains with two K15 alleles, P and M. We found that the PLCγ2 cSH2 domain, by binding to K15P, can be used as dominant negative inhibitor of the K15P-PLCγ1 interaction, K15P-dependent PLCγ1 phosphorylation, NFAT-dependent promoter activation and the increased invasiveness and angiogenic properties of KSHV infected endothelial cells. We increased the binding of the PLCγ2 cSH2 domain for K15P by substituting two amino acids, thereby creating an improved dominant negative inhibitor of the K15P-dependent PLCγ1 activation. Taken together, these results demonstrate a necessary role of K15 in the increased invasiveness and angiogenesis of KSHV infected endothelial cells and suggest the K15-PLCγ1 interaction as a possible new target for inhibiting the angiogenic and invasive properties of KSHV. AU - Gramolelli, S.* AU - Weidner-Glunde, M.* AU - Abere, B.* AU - Viejo-Borbolla, A.* AU - Bala, K.* AU - Rückert, J.* AU - Kremmer, E. AU - Schulz, T.F.* C1 - 46610 C2 - 37663 CY - San Francisco TI - Inhibiting the recruitment of PLCγ1 to Kaposi's Sarcoma Herpesvirus K15 protein reduces the invasiveness and angiogenesis of infected endothelial cells. JO - PLoS Pathog. VL - 11 IS - 8 PB - Public Library Science PY - 2015 SN - 1553-7366 ER - TY - JOUR AB - Herpesviruses form different gH/gL virion envelope glycoprotein complexes that serve as entry complexes for mediating viral cell-type tropism in vitro; their roles in vivo, however, remained speculative and can be addressed experimentally only in animal models. For murine cytomegalovirus two alternative gH/gL complexes, gH/gL/gO and gH/gL/MCK-2, have been identified. A limitation of studies on viral tropism in vivo has been the difficulty in distinguishing between infection initiation by viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. As a new strategy to dissect these two events, we used a gO-transcomplemented ΔgO mutant for providing the gH/gL/gO complex selectively for the initial entry step, while progeny virions lack gO in subsequent rounds of infection. Whereas gH/gL/gO proved to be critical for establishing infection by efficient entry into diverse cell types, including liver macrophages, endothelial cells, and hepatocytes, it was dispensable for intra-tissue spread. Notably, the salivary glands, the source of virus for host-to-host transmission, represent an exception in that entry into virus-producing cells did not strictly depend on either the gH/gL/gO or the gH/gL/MCK-2 complex. Only if both complexes were absent in gO and MCK-2 double-knockout virus, in vivo infection was abolished at all sites. AU - Lemmermann, N.A.W.* AU - Krmpotic, A.* AU - Podlech, J.* AU - Brizic, I.* AU - Prager, A.* AU - Adler, H. AU - Karbach, A.* AU - Wu, Y.* AU - Jonjic, S.* AU - Reddehase, M.J.* AU - Adler, B.* C1 - 43235 C2 - 36281 CY - San Francisco TI - Non-redundant and redundant roles of cytomegalovirus gH/gL complexes in host organ entry and intra-tissue spread. JO - PLoS Pathog. VL - 11 IS - 2 PB - Public Library Science PY - 2015 SN - 1553-7366 ER - TY - JOUR AB - The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects the majority of the human population. All EBV strains transform B lymphocytes, but some strains, such as M81, also induce spontaneous virus replication. EBV encodes 22 microRNAs (miRNAs) that form a cluster within the BART region of the virus and have been previously been found to stimulate tumor cell growth. Here we describe their functions in B cells infected by M81. We found that the BART miRNAs are downregulated in replicating cells, and that exposure of B cells in vitro or in vivo in humanized mice to a BART miRNA knockout virus resulted in an increased proportion of spontaneously replicating cells, relative to wild type virus. The BART miRNAs subcluster 1, and to a lesser extent subcluster 2, prevented expression of BZLF1, the key protein for initiation of lytic replication. Thus, multiple BART miRNAs cooperate to repress lytic replication. The BART miRNAs also downregulated pro- and anti-apoptotic mediators such as caspase 3 and LMP1, and their deletion did not sensitize B-cells to apoptosis. To the contrary, the majority of humanized mice infected with the BART miRNA knockout mutant developed tumors more rapidly, probably due to enhanced LMP1 expression, although deletion of the BART miRNAs did not modify the virus transforming abilities in vitro. This ability to slow cell growth could be confirmed in non-humanized immunocompromized mice. Injection of resting B cells exposed to a virus that lacks the BART miRNAs resulted in accelerated tumor growth, relative to wild type controls. Therefore, we found that the M81 BART miRNAs do not enhance B-cell tumorigenesis but rather repress it. The repressive effects of the BART miRNAs on potentially pathogenic viral functions in infected B cells are likely to facilitate long-term persistence of the virus in the infected host. AU - Lin, X.* AU - Tsai, M.H.* AU - Shumilov, A.* AU - Poirey, R.* AU - Bannert, H.* AU - Middeldorp, J.M.* AU - Feederle, R. AU - Delecluse, H.J.* C1 - 47651 C2 - 39377 TI - The Epstein-Barr virus BART miRNA cluster of the M81 strain modulates multiple functions in primary B cells. JO - PLoS Pathog. VL - 11 IS - 12 PY - 2015 SN - 1553-7366 ER - TY - JOUR AB - The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms. AU - Rancan, C. AU - Schirrmann, L. AU - Hüls, C. AU - Zeidler, R. AU - Moosmann, A. C1 - 45183 C2 - 37259 CY - San Francisco TI - Latent membrane protein LMP2A impairs recognition of EBV-infected cells by CD8+ T cells. JO - PLoS Pathog. VL - 11 IS - 6 PB - Public Library Science PY - 2015 SN - 1553-7366 ER - TY - JOUR AB - Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies. AU - Linnerbauer, S. AU - Behrends, U. AU - Adhikary, D. AU - Witter, K.* AU - Bornkamm, G.W. AU - Mautner, J. C1 - 31373 C2 - 34493 CY - San Francisco TI - Virus and autoantigen-specific CD4+ T cells are key effectors in a SCID mouse model of EBV-associated post-transplant lymphoproliferative disorders. JO - PLoS Pathog. VL - 10 IS - 5 PB - Public Library Science PY - 2014 SN - 1553-7366 ER - TY - JOUR AB - Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles. AU - Ma, H.C.* AU - Liu, Y.* AU - Wang, C.* AU - Strauss, M.* AU - Rehage, N. AU - Chen, Y.H.* AU - Altan-Bonnet, N.* AU - Hogle, J.* AU - Wimmer, E.* AU - Mueller, S.* AU - Paul, A.V.* AU - Jiang, P.* C1 - 31050 C2 - 34151 TI - An interaction between glutathione and the capsid is required for the morphogenesis of c-cluster enteroviruses. JO - PLoS Pathog. VL - 10 IS - 4 PY - 2014 SN - 1553-7366 ER - TY - JOUR AB - JC polyomavirus (JCV) carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML) which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV mark infection occur only in 50-60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA), instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate for JCV infection, were compared to HLA class I and II alleles in 1621 Scandinavian persons with MS and 1064 population-based Swedish controls and associations were replicated in 718 German persons with MS. HLA-alleles were determined by SNP imputation, sequence specific (SSP) kits and a reverse PCR sequence-specific oligonucleotide (PCR-SSO) method. An initial GWAS screen displayed a strong HLA class II region signal. The HLA-DRB1*15 haplotype was strongly negatively associated to JCV sero-status in Scandinavian MS cases (OR = 0.42, p = 7×10-15) and controls (OR = 0.53, p = 2×10-5). In contrast, the DQB1*06:03 haplotype was positively associated with JCV sero-status, in Scandinavian MS cases (OR = 1.63, p = 0.006), and controls (OR = 2.69, p = 1×10-5). The German dataset confirmed these findings (OR = 0.54, p = 1×10-4 and OR = 1.58, p = 0.03 respectively for these haplotypes). HLA class II restricted immune responses, and hence CD4+ T cell immunity is pivotal for JCV infection control. Alleles within the HLA-DR1*15 haplotype are associated with a protective effect on JCV infection. Alleles within the DQB1*06:03 haplotype show an opposite association. These associations between JC virus antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and lays the ground for risk stratification for PML and development of therapy and prevention. AU - Sundqvist, E.* AU - Buck, D.* AU - Warnke, C.* AU - Albrecht, E. AU - Gieger, C. AU - Khademi, M.* AU - Lima Bomfim, I.* AU - Fogdell-Hahn, A.* AU - Link, J.* AU - Alfredsson, L.* AU - Søndergaard, H.B.* AU - Hillert, J.* AU - Oturai, A.B.* AU - Hemme, B.* AU - Kockum, I.* AU - Olsson, T.* C1 - 31114 C2 - 34142 TI - JC polyomavirus infection is strongly controlled by human leucocyte antigen class II variants. JO - PLoS Pathog. VL - 10 IS - 4 PY - 2014 SN - 1553-7366 ER - TY - JOUR AB - Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts. AU - van Gent, M.* AU - Braem, S.G.* AU - de Jong, A.* AU - Delagic, N.* AU - Peeters, J.G.* AU - Boer, I.G.* AU - Moynagh, P.N.* AU - Kremmer, E. AU - Wiertz, E.J.* AU - Ovaa, H.* AU - Griffin, B.D.* AU - Ressing, M.E.* C1 - 30841 C2 - 33946 CY - San Francisco TI - Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling. JO - PLoS Pathog. VL - 10 IS - 2 PB - Public Library Science PY - 2014 SN - 1553-7366 ER - TY - JOUR AB - Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy. AU - Ameres, S. AU - Mautner, J. AU - Schlott, F. AU - Neuenhahn, M. AU - Busch, D.H. AU - Plachter, B.* AU - Moosmann, A. C1 - 25758 C2 - 31907 TI - Presentation of an immunodominant immediate-early CD8+ T cell epitope resists human cytomegalovirus immunoevasion. JO - PLoS Pathog. VL - 9 IS - 5 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies. AU - Ching, W.* AU - Koyuncu, E.* AU - Singh, S.* AU - Arbelo-Roman, C.* AU - Härtl, B.* AU - Kremmer, E. AU - Speiseder, T.* AU - Meier, C.* AU - Dobner, T.* C1 - 24246 C2 - 31356 TI - A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation. JO - PLoS Pathog. VL - 9 IS - 3 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing gene expression profiles of wt and EBNA3A negative EBV infected B cells, we have previously identified a broad array of cellular genes controlled by EBNA3A. We now find that genes repressed by EBNA3A in these cells are significantly enriched for the repressive histone mark H3K27me3, which is installed by Polycomb group (PcG) proteins. This PcG-controlled subset of genes also carries H3K27me3 marks in a variety of other tissues, suggesting that the commitment to PcG silencing is an intrinsic feature of these gene loci that can be used by EBNA3A. In addition, EBNA3A targets frequently reside in co-regulated gene clusters. To study the mechanism of gene repression by EBNA3A and to evaluate the relative contribution of PcG proteins during this process, we have selected the genomic neighbors CXCL10 and CXCL9 as a model for co-repressed and PcG-controlled genes. We show that EBNA3A binds to CBF1 occupied intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes in a CBF1-dependent manner and initiation of a delayed gain of H3K27me3 marks covering an extended chromatin domain. H3K27me3 marks increase gradually and are maintained by EBNA3A. Our study provides direct evidence that repression by EBNA3A requires CBF1 and that EBNA3A and EBNA2 compete for access to CBF1 at identical genomic sites. Most importantly, our results demonstrate that transcriptional silencing by EBNA3A precedes the appearance of repressive PcG marks and indicate that both events are triggered by loss of enhancer activity. AU - Harth-Hertle, M.L. AU - Scholz, B.A. AU - Erhard, F.* AU - Glaser, L.V. AU - Dölken, L.* AU - Zimmer, R.* AU - Kempkes, B. C1 - 27782 C2 - 32810 TI - Inactivation of intergenic enhancers by EBNA3A initiates and maintains polycomb signatures across a chromatin domain encoding CXCL10 and CXCL9. JO - PLoS Pathog. VL - 9 IS - 9 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed 'LANA speckles', which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA 'nuclear speckles' and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence. AU - Hellert, J.* AU - Weidner-Glunde, M.* AU - Krausze, J.* AU - Richter, U.* AU - Adler, H. AU - Fedorov, R.* AU - Pietrek, M.* AU - Rückert, J.* AU - Ritter, C.* AU - Schulz, T.F.* AU - Lührs, T.* C1 - 27865 C2 - 32836 CY - San Francisco TI - A structural basis for BRD2/4-mediated host chromatin interaction and oligomer assembly of Kaposi sarcoma-associated herpesvirus and murine gammaherpesvirus LANA proteins. JO - PLoS Pathog. VL - 9 IS - 10 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors. AU - McClellan, M.J.* AU - Wood, C.D.* AU - Ojeniyi, O.* AU - Cooper, T.J.* AU - Kanhere, A.* AU - Arvey, A.* AU - Webb, H.M.* AU - Palermo, R.D.* AU - Harth-Hertle, M.L. AU - Kempkes, B. AU - Jenner, R.G.* AU - West, M.J.* C1 - 27780 C2 - 32809 TI - Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming. JO - PLoS Pathog. VL - 9 IS - 9 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade. AU - Scholz, B.A. AU - Harth-Hertle, M.L. AU - Malterer, G.* AU - Haas, J.* AU - Ellwart, J. AU - Schulz, T.F.* AU - Kempkes, B. C1 - 25762 C2 - 31906 TI - Abortive lytic reactivation of KSHV in CBF1/CSL deficient human B cell lines. JO - PLoS Pathog. VL - 9 IS - 5 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from C-13-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity. AU - Sixt, B.S.* AU - Siegl, A.* AU - Müller, C. AU - Watzka, M.* AU - Wultsch, A.* AU - Tziotis, D. AU - Montanaro, J.* AU - Richter, A.* AU - Schmitt-Kopplin, P. AU - Horn, M.* C1 - 27543 C2 - 32714 TI - Metabolic features of Protochlamydia amoebophila elementary bodies - a link between activity and infectivity in Chlamydiae. JO - PLoS Pathog. VL - 9 IS - 8 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8(+) T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming "nodular inflammatory foci" (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control. AU - Stahl, F.R.* AU - Heller, K.* AU - Halle, S.* AU - Keyser, K.A.* AU - Busche, A.* AU - Marquardt, A.* AU - Wagner, K.* AU - Boelter, J.* AU - Bischoff, Y.* AU - Kremmer, E. AU - Arens, R.* AU - Messerle, M.* AU - Forster, R.* C1 - 28885 C2 - 33562 CY - San Francisco TI - Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung. JO - PLoS Pathog. VL - 9 IS - 12 PB - Public Library Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination. AU - Wagner, F.M.* AU - Brizic, I.* AU - Prager, A.* AU - Trsan, T.* AU - Arapovic, M.* AU - Lemmermann, N.A.W.* AU - Podlech, J.* AU - Reddehase, M.J.* AU - Lemnitzer, F.* AU - Bosse, J.B.* AU - Gimpfl, M.* AU - Marcinowski, L.* AU - MacDonald, M.* AU - Adler, H. AU - Koszinowski, U.H.* AU - Adler, B.* C1 - 26068 C2 - 32053 TI - The viral chemokine MCK-2 of murine cytomegalovirus promotes infection as part of a gH/gL/MCK-2 complex. JO - PLoS Pathog. VL - 9 IS - 7 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen. AU - Wiemann, P.* AU - Sieber, C.M.K. AU - von Bargen, K.W.* AU - Studt, L.* AU - Niehaus, E.-M.* AU - Espino, J.J.* AU - Huß, K.* AU - Michielse, C.B.* AU - Albermann, S.* AU - Wagner, D.E.* AU - Bergner, S.V.* AU - Connolly, L.R.* AU - Fischer, A.* AU - Reuter, G.* AU - Kleigrewe, K.* AU - Bald, T.* AU - Wingfield, B.D.* AU - Ophir, R.* AU - Freeman, S.* AU - Hippler, M.* AU - Smith, K.M.* AU - Brown, D.W.* AU - Proctor, R.H.* AU - Münsterkötter, M. AU - Freitag, M.* AU - Humpf, H.-U.* AU - Güldener, U. AU - Tudzynski, B.* C1 - 25162 C2 - 31837 TI - Deciphering the cryptic genome: Genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. JO - PLoS Pathog. VL - 9 IS - 6 PB - Public Library of Science PY - 2013 SN - 1553-7366 ER - TY - JOUR AB - Lifelong persistence of Epstein-Barr virus (EBV) in infected hosts is mainly owed to the virus' pronounced abilities to evade immune responses of its human host. Active immune evasion mechanisms reduce the immunogenicity of infected cells and are known to be of major importance during lytic infection. The EBV genes BCRF1 and BNLF2a encode the viral homologue of IL-10 (vIL-10) and an inhibitor of the transporter associated with antigen processing (TAP), respectively. Both are known immunoevasins in EBV's lytic phase. Here we describe that BCRF1 and BNLF2a are functionally expressed instantly upon infection of primary B cells. Using EBV mutants deficient in BCRF1 and BNLF2a, we show that both factors contribute to evading EBV-specific immune responses during the earliest phase of infection. vIL-10 impairs NK cell mediated killing of infected B cells, interferes with CD4+ T-cell activity, and modulates cytokine responses, while BNLF2a reduces antigen presentation and recognition of newly infected cells by EBV-specific CD8+ T cells. Together, both factors significantly diminish the immunogenicity of EBV-infected cells during the initial, pre-latent phase of infection and may improve the establishment of a latent EBV infection in vivo. AU - Jochum, S. AU - Moosmann, A. AU - Lang, S.* AU - Hammerschmidt, W. AU - Zeidler, R.* C1 - 11600 C2 - 30709 TI - The EBV immunoevasins vIL-10 and BNLF2a protect newly infected B cells from immune recognition and elimination. JO - PLoS Pathog. VL - 8 IS - 5 PB - Public Library of Science PY - 2012 SN - 1553-7366 ER - TY - JOUR AB - Epigenetic mechanisms are essential for the regulation of all genes in mammalian cells but transcriptional repression including DNA methylation are also major epigenetic mechanisms of defense inactivating potentially harmful pathogens. Epstein-Barr Virus (EBV), however, has evolved to take advantage of CpG methylated DNA to regulate its own biphasic life cycle. We show here that latent EBV DNA has an extreme composition of methylated CpG dinucleotides with a bimodal distribution of unmethylated or fully methylated DNA at active latent genes or completely repressed lytic promoters, respectively. We find this scenario confirmed in primary EBV-infected memory B cells in vivo. Extensive CpG methylation of EBV's DNA argues for a very restricted gene expression during latency. Above-average nucleosomal occupancy, repressive histone marks, and Polycomb-mediated epigenetic silencing further shield early lytic promoters from activation during latency. The very tight repression of viral lytic genes must be overcome when latent EBV enters its lytic phase and supports de novo virus synthesis in infected cells. The EBV-encoded and AP-1 related transcription factor BZLF1 overturns latency and initiates virus synthesis in latently infected cells. Paradoxically, BZLF1 preferentially binds to CpG-methylated motifs in key viral promoters for their activation. Upon BZLF1 binding, we find nucleosomes removed, Polycomb repression lost, and RNA polymerase II recruited to the activated early promoters promoting efficient lytic viral gene expression. Surprisingly, DNA methylation is maintained throughout this phase of viral reactivation and is no hindrance to active transcription of extensively CpG methylated viral genes as thought previously. Thus, we identify BZLF1 as a pioneer factor that reverses epigenetic silencing of viral DNA to allow escape from latency and report on a new paradigm of gene regulation. AU - Wöllmer, A. AU - Arteaga-Salas, J.M.* AU - Hammerschmidt, W. C1 - 10889 C2 - 30436 TI - BZLF1 governs CpG-methylated chromatin of Epstein-Barr virus reversing epigenetic repression. JO - PLoS Pathog. VL - 8 IS - 9 PB - Public Library of Science PY - 2012 SN - 1553-7366 ER - TY - JOUR AB - Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVAΔF1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVAΔF1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVAΔF1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling. AU - Eitz Ferrer, P.* AU - Potthoff, S.* AU - Kirschnek, S.* AU - Gasteiger, G. AU - Kastenmüller, W. AU - Ludwig, H.* AU - Paschen, S.A.* AU - Villunger, A.* AU - Sutter, G.* AU - Drexler, I. AU - Häcker, G.* C1 - 6397 C2 - 28613 TI - Induction of noxa-mediated apoptosis by modified vaccinia virus Ankara depends on viral recognition by cytosolic helicases, leading to IRF-3/IFN-β-dependent induction of pro-apoptotic noxa. JO - PLoS Pathog. VL - 7 IS - 6 PB - Public Library of Science PY - 2011 SN - 1553-7366 ER - TY - JOUR AB - Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead barley roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyle strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarray analysis, argues for a biphasic root colonization strategy of P. indica. This is supported by a cytological study that shows an early biotrophic growth followed by a cell death-associated phase. About 10% of the fungal genes induced during the biotrophic colonization encoded putative small secreted proteins (SSP), including several lectin-like proteins and members of a P. indica-specific gene family (DELD) with a conserved novel seven-amino acids motif at the C-terminus. Similar to effectors found in other filamentous organisms, the occurrence of the DELDs correlated with the presence of transposable elements in gene-poor repeat-rich regions of the genome. This is the first in depth genomic study describing a mutualistic symbiont with a biphasic lifestyle. Our findings provide a significant advance in understanding development of biotrophic plant symbionts and suggest a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi. AU - Zuccaro, A.* AU - Lahrmann, U.* AU - Güldener, U. AU - Langen, G.* AU - Pfiffi, S.* AU - Biedenkopf, D.* AU - Wong, P. AU - Samans, B.* AU - Grimm, C.* AU - Basiewicz, M.* AU - Murat, C.* AU - Martin, F.* AU - Kogel, K.H.* C1 - 6929 C2 - 29436 TI - Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont Piriformospora indica. JO - PLoS Pathog. VL - 7 IS - 10 PB - Public Library of Science PY - 2011 SN - 1553-7366 ER - TY - JOUR AB - DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to 'read' DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing. AU - Bergbauer, M. AU - Kalla, M. AU - Schmeinck, A. AU - Göbel, C. AU - Rothbauer, U.* AU - Eck, S. AU - Benet-Pagès, A. AU - Strom, T.M. AU - Hammerschmidt, W. C1 - 5736 C2 - 27563 SP - e1001114 TI - CpG-methylation regulates a class of Epstein-Barr virus promoters. JO - PLoS Pathog. VL - 6 IS - 9 PB - Public Library of Science PY - 2010 SN - 1553-7366 ER - TY - JOUR AB - We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts. AU - Parameswaran, P.* AU - Sklan, E.* AU - Wilkins, C.* AU - Burgon, T.* AU - Samuel, M.A.* AU - Lu, R.* AU - Ansel, K.M.* AU - Heissmeyer, V. AU - Einav, S.* AU - Jackson, W.* AU - Douka, T.* AU - Paranjape, S.* AU - Polacek, C.* AU - dos Santos, F.B.* AU - Jalili, R.* AU - Babrzadeh, F. AU - Gharizadeh, B.* AU - Grimm, D.* AU - Kay, M.* AU - Koike, S.* AU - Sarnow, P.* AU - Ronaghi, M.* AU - Ding, S.W.* AU - Harris, E.* AU - Chow, M.* AU - Diamond, M.S.* AU - Kirkegaard, K.* AU - Glenn, J.S.* AU - Fire, A.Z.* C1 - 194 C2 - 27062 TI - Six RNA viruses and forty-one hosts: Viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems. JO - PLoS Pathog. VL - 6 IS - 2 PB - Public Library Science PY - 2010 SN - 1553-7366 ER - TY - JOUR AB - Cellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase. AU - Seto, E. AU - Moosmann, A. AU - Grömminger, S. AU - Walz, N.* AU - Grundhoff, A.* AU - Hammerschmidt, W. C1 - 5764 C2 - 27725 SP - 69-70 TI - Micro RNAs of Epstein-Barr virus promote cell cycle progression and prevent apoptosis of primary human B cells. JO - PLoS Pathog. VL - 6 IS - 8 PB - Public Library of Science PY - 2010 SN - 1553-7366 ER - TY - JOUR AB - The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine- learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity oF similar to 71% and selectivity of similar to 85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will facilitate further studies on and improve our understanding of type III secretion and its role in pathogen-host interactions. AU - Arnold, R.* AU - Brandmaier, S.* AU - Kleine, F.* AU - Tischler, P.* AU - Heinz, E. AU - Behrens, S.* AU - Niinikoski, A.* AU - Mewes, H.-W. AU - Horn, M.* AU - Rattei, T.* C1 - 225 C2 - 27011 TI - Sequence-based prediction of type III secreted proteins. JO - PLoS Pathog. VL - 5 IS - 4 PB - Public Library of Science PY - 2009 SN - 1553-7366 ER - TY - JOUR AB - The gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8(+) T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide-and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8(+) T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (Delta BNLF2a) and compared the ability of Delta BNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8(+) T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by DBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8(+) T cells remained equally poor when presented by both wildtype and Delta BNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet Delta BNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle. AU - Croft, N.P.* AU - Shannon-Lowe, C.* AU - Bell, A.I.* AU - Horst, D.* AU - Kremmer, E. AU - Ressing, M.E.* AU - Wiertz, E.J.* AU - Middeldorp, J.M.* AU - Rowe, M.* AU - Rickinson, A.B.* AU - Hislop, A.D.* C1 - 986 C2 - 26507 TI - Stage-specific inhibition of MHC Class I presentation by the Epstein-Barr virus BNLF2a protein during virus lytic cycle. JO - PLoS Pathog. VL - 5 IS - 6 PB - Public Library of Science PY - 2009 SN - 1553-7366 ER - TY - JOUR AB - The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV-growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis. AU - Hertle, M.L. AU - Popp, C. AU - Petermann, S. AU - Maier, S. AU - Kremmer, E. AU - Lang, R.* AU - Mages, J.* AU - Kempkes, B. C1 - 827 C2 - 26604 TI - Differential gene expression patterns of EBV infected EBNA-3A positive and negative human B lymphocytes. JO - PLoS Pathog. VL - 5 IS - 7 PB - Public Library of Science PY - 2009 SN - 1553-7366 ER - TY - JOUR AB - Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth-transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc-driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro-transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo. AU - Kelly, G.L. AU - Long, H.M.* AU - Stylianou, J.* AU - Thomas, W.A.* AU - Leese, A.* AU - Bell, A.I.* AU - Bornkamm, G.W. AU - Mautner, J.* AU - Rickinson, A.B.* AU - Rowe, M.* C1 - 1458 C2 - 26966 TI - An Epstein-Barr virus anti-apoptotic protein constitutively expressed in transformed cells and implicated in burkitt lymphomagenesis: The Wp/BHRF1 link. JO - PLoS Pathog. VL - 5 IS - 3 PB - Public Library of Science PY - 2009 SN - 1553-7366 ER -