TY - JOUR AB - Apobec3A is involved in the antiviral host defense, targeting nuclear DNA, introducing point mutations, and thereby activating DNA damage response (DDR). Here, we found a significant upregulation of Apobec3A during HAdV infection, including Apobec3A protein stabilization mediated by the viral proteins E1B-55K and E4orf6, which subsequently limited HAdV replication and most likely involved a deaminase-dependent mechanism. The transient silencing of Apobec3A enhanced adenoviral replication. HAdV triggered Apobec3A dimer formation and enhanced activity to repress the virus. Apobec3A decreased E2A SUMOylation and interfered with viral replication centers. A comparative sequence analysis revealed that HAdV types A, C, and F may have evolved a strategy to escape Apobec3A-mediated deamination via reduced frequencies of TC dinucleotides within the viral genome. Although viral components induce major changes within infected cells to support lytic life cycles, our findings demonstrate that host Apobec3A-mediated restriction limits virus replication, albeit that HAdV may have evolved to escape this restriction. This allows for novel insights into the HAdV/host-cell interplay, which broaden the current view of how a host cell can limit HAdV infection. IMPORTANCE Our data provide a novel conceptual insight into the virus/host-cell interplay, changing the current view of how a host-cell can defeat a virus infection. Thus, our study reveals a novel and general impact of cellular Apobec3A on the intervention of human adenovirus (HAdV) gene expression and replication by improving the host antiviral defense mechanisms, thereby providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways that are modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as COVID vaccine vectors and also frequently serve as tools in human gene therapy and oncolytic treatment options. HAdV constitute an ideal model system by which to analyze the transforming capabilities of DNA tumor viruses as well as the underlying molecular principles of virus-induced and cellular tumorigenesis. AU - Göttig, L.* AU - Weiß, C.* AU - Stubbe, M.* AU - Hanrieder, L.* AU - Hofmann, S.* AU - Grodziecki, A.* AU - Stadler, D.* AU - Carpentier, A.* AU - Protzer, U. AU - Schreiner, S. C1 - 67763 C2 - 54241 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Apobec3A deamination functions are involved in antagonizing efficient human adenovirus replication and gene expression. JO - mBio VL - 14 IS - 3 PB - Amer Soc Microbiology PY - 2023 SN - 2150-7511 ER - TY - JOUR AB - Eosinophilia is associated with various persisting inflammatory diseases and often coincides with chronic fungal infections or fungal allergy as in the case of allergic bronchopulmonary aspergillosis (ABPA). Here, we show that intranasal administration of live Aspergillus fumigatus conidia causes fatal lung damage in eosinophilic interleukin-5 (IL-5)-transgenic mice. To further investigate the activation of eosinophils by A. fumigatus, we established a coculture system of mouse bone marrow-derived eosinophils (BMDE) with different A. fumigatus morphotypes and analyzed the secretion of cytokines, chemokines, and eicosanoids. A. fumigatus-stimulated BMDE upregulated expression of CD11b and downregulated CD62L and CCR3. They further secreted several proinflammatory mediators, including IL-4, IL-13, IL-18, macrophage inflammatory protein-1α (MIP-1α)/CC chemokine ligand 3 (CCL3), MIP-1β/CCL4, and thromboxane. This effect required direct interaction and adherence between eosinophils and A. fumigatus, as A. fumigatus culture supernatants or A. fumigatus mutant strains with impaired adhesion elicited a rather poor eosinophil response. Unexpectedly, canonical Toll-like receptor (TLR) or C-type-lectin receptor (CLR) signaling was largely dispensable, as the absence of MYD88, TRIF, or caspase recruitment domain-containing protein 9 (CARD9) resulted in only minor alterations. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway in A. fumigatus-induced eosinophil activation. Correspondingly, we could show that phosphatidylinositol 3-kinase (PI3K) inhibitors successfully prevent A. fumigatus-induced eosinophil activation. The PI3K pathway in eosinophils may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders. IMPORTANCE Allergic bronchopulmonary aspergillosis (ABPA) is caused by the fungus Aspergillus fumigatus, afflicts about five million patients globally, and is still a noncurable disease. ABPA is associated with pronounced lung eosinophilia. Activated eosinophils enhance the inflammatory response not only by degranulation of toxic proteins but also by secretion of small effector molecules. Receptors and signaling pathways involved in activation of eosinophils by A. fumigatus are currently unknown. Here, we show that A. fumigatus-elicited activation of eosinophils requires direct cell-cell contact and results in modulation of cell surface markers and rapid secretion of cytokines, chemokines, and lipid mediators. Unexpectedly, this activation occurred independently of canonical Toll-like receptor or C-type lectin receptor signaling. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway, and PI3K inhibitors successfully prevented A. fumigatus-induced eosinophil activation. The PI3K pathway may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders. AU - Dietschmann, A.* AU - Schruefer, S.* AU - Westermann, S.* AU - Henkel, F. AU - Castiglione, K.* AU - Willebrand, R.* AU - Adam, J.* AU - Ruland, J.* AU - Lang, R.* AU - Sheppard, D.C.* AU - Esser-von Bieren, J. AU - Radtke, D.* AU - Krappmann, S.* AU - Voehringer, D.* C1 - 65435 C2 - 52679 TI - Phosphatidylinositol 3-kinase (PI3K) orchestrates Aspergillus fumigatus-induced eosinophil activation independently of canonical toll-like receptor (TLR)/C-type-lectin receptor (CLR) signaling. JO - mBio VL - 13 IS - 4 PY - 2022 SN - 2150-7511 ER - TY - JOUR AB - Manipulating soil metabolism through heavy inoculation with microbes is feasible if organic wastes can be utilized as the substrate for growth and vector as a fertilizer. This, however, requires organisms active in both digestate and soil (generalists). Here, we present a dual enrichment strategy to enrich and isolate such generalists among N2O-respiring bacteria (NRB) in soil and digestates, to be used as an inoculum for strengthening the N2O-reduction capacity of soils. The enrichment strategy utilizes sequential batch enrichment cultures alternating between sterilized digestate and soil as substrates, with each batch initiated with limited O2 and unlimited N2O. The cultures were monitored for gas kinetics and community composition. As predicted by a Lotka-Volterra competition model, cluster analysis identified generalist operational taxonomic units (OTUs) which became dominant, digestate/soil-specialists which did not, and a majority that were gradually diluted out. We isolated several NRBs circumscribed by generalist OTUs. Their denitrification genes and phenotypes predicted a variable capacity to act as N2O-sinks, while all genomes predicted broad catabolic capacity. The latter contrasts with previous attempts to enrich NRB by anaerobic incubation of unsterilized digestate only, which selected for organisms with a catabolic capacity limited to fermentation products. The two isolates with the most promising characteristics as N2O sinks were a Pseudomonas sp. with a full-fledged denitrification-pathway and a Cloacibacterium sp. carrying only N2O reductase (clade II), and soil experiments confirmed their capacity to reduce N2O-emissions from soil. The successful enrichment of NRB with broad catabolic spectra suggests that the concept of dual enrichment should also be applicable for enrichment of generalists with traits other than N2O reduction. IMPORTANCE N2O emissions from farmed soils are a major source of climate forcing. Here, denitrifying bacteria act as both source and sink for N2O, determined by regulatory traits or the absence of genes coding for the enzymes producing or reducing N2O. One approach to reducing emissions is to amend large numbers of N2O-reducing bacteria (NRB) to soil. This was shown to be feasible by growing NRB to high densities in organic wastes and then applying them as fertilizers. The effect on N2O emissions, however, was transient because the isolated NRBs were unsuited to soil. Here, we have developed an enrichment strategy selecting for organisms with generalist lifestyles, tolerant of rapid environmental changes. This was used to isolate robust NRBs that grow both in digestate and when amended to soils. This strategy opens an avenue for obtaining not just robust NRBs to reduce N2O emissions, but any organism destined for application to complex environments. AU - Jonassen, K.R.* AU - Ormasen, I.* AU - Duffner, C. AU - Hvidsten, T.R.* AU - Bakken, L.R.* AU - Vick, S.H.W.* C1 - 65398 C2 - 52297 TI - A dual enrichment strategy provides soil- and digestate-competent nitrous oxide-respiring bacteria for mitigating climate forcing in agriculture. JO - mBio VL - 13 IS - 3 PY - 2022 SN - 2150-7511 ER - TY - JOUR AB - Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plas-macytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV. IMPORTANCE Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/β receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune-evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs of Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the antiviral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN-α and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection. AU - Bouvet, M. AU - Voigt, S. AU - Tagawa, T. AU - Albanese, M. AU - Chen, Y.-F. A. AU - Chen, Y.* AU - Fachko, D.N.* AU - Pich, D. AU - Göbel, C. AU - Skalsky, R.L.* AU - Hammerschmidt, W. C1 - 61723 C2 - 50138 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Multiple viral micrornas regulate interferon release and signaling early during infection with epstein-barr virus. JO - mBio VL - 12 IS - 2 PB - Amer Soc Microbiology PY - 2021 SN - 2150-7511 ER - TY - JOUR AB - The innate immune system serves as frontline defense against pathogens, such as bacteria and viruses. Natural killer (NK) cells are a part of innate immunity and can both secrete cytokines and directly target cells for lysis. NK cells express several cell surface receptors, including NKG2D, which bind multiple ligands. People with deficiencies in NK cells are often susceptible to uncontrolled infection by herpesviruses, such as Epstein-Barr virus (EBV). Infection with EBV stimulates both innate and adaptive immunity, yet the virus establishes lifelong latent infection in memory B cells. We show that the EBV oncogene EBNA1, previously known to be necessary for maintaining EBV genomes in latently infected cells, also plays an important role in suppressing NK cell responses and cell death in newly infected cells. EBNA1 does so by downregulating the NKG2D ligands ULBP1 and ULBP5 and modulating expression of c-Myc. B cells infected with a derivative of EBV that lacks EBNA1 are more susceptible to NK cell-mediated killing and show increased levels of apoptosis. Thus, EBNA1 performs a previously unappreciated role in reducing immune response and programmed cell death after EBV infection, helping infected cells avoid immune surveillance and apoptosis and thus persist for the lifetime of the host. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous human pathogen, infecting up to 95% of the world's adult population. Initial infection with EBV can cause infectious mononucleosis. EBV is also linked to several human malignancies, including lymphomas and carcinomas. Although infection by EBV alerts the immune system and causes an immune response, the virus persists for life in memory B cells. We show that the EBV protein EBNA1 can downregulate several components of the innate immune system linked to natural killer (NK) cells. This downregulation of NK cell activity translates to lower killing of EBV-infected cells and is likely one way that EBV escapes immune surveillance after infection. Additionally, we show that EBNA1 reduces apoptosis in newly infected B cells, allowing more of these cells to survive. Taken together, our findings uncover new functions of EBNA1 and provide insights into viral strategies to survive the initial immune response postinfection. AU - Westhoff Smith, D.* AU - Chakravorty, A.* AU - Hayes, M.* AU - Hammerschmidt, W. AU - Sugden, B.* C1 - 63650 C2 - 51741 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - The Epstein-Barr virus oncogene EBNA1 suppresses natural killer cell responses and apoptosis early after infection of peripheral B cells. JO - mBio VL - 12 IS - 6 PB - Amer Soc Microbiology PY - 2021 SN - 2150-7511 ER - TY - JOUR AB - Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneurnophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages.IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-1, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila. AU - Herkt, C.E.* AU - Caffrey, B.E.* AU - Surmann, K.* AU - Blankenburg, S.* AU - Gesell Salazar, M.* AU - Jung, A.L.* AU - Herbel, S.M.* AU - Hoffmann, K.* AU - Schulte, L.N.* AU - Chen, W.* AU - Sittka-Stark, A.* AU - Völker, U.* AU - Vingron, M.* AU - Marsico, A. AU - Bertrams, W.* AU - Schmeck, B.* C1 - 58712 C2 - 48606 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - A miRNA network controls Legionella pneumophila replication in human macrophages via LGALS8 and MX1. JO - mBio VL - 11 IS - 2 PB - Amer Soc Microbiology PY - 2020 SN - 2150-7511 ER - TY - JOUR AB - Colicins are toxins produced and released by Enterobacteriaceae to kill competitors in the gut. While group A colicins employ a division of labor strategy to liberate the toxin into the environment via colicin-specific lysis, group B colicin systems lack cognate lysis genes. In Salmonella enterica serovar Typhimurium (S. Tm), the group B colicin lb (Collb) is released by temperate phage-mediated bacteriolysis. Phage-mediated Collb release promotes S. Tm fitness against competing Escherichia coll. It remained unclear how prophage-mediated lysis is realized in a clonal population of Collb producers and if prophages contribute to evolutionary stability of toxin release in S. Tm. Here, we show that prophage-mediated lysis occurs in an S. Tm subpopulation only, thereby introducing phenotypic heterogeneity to the system. We established a mathematical model to study the dynamic interplay of S. Tm, Collb, and a temperate phage in the presence of a competing species. Using this model, we studied long-term evolution of phage lysis rates in a fluctuating infection scenario. This revealed that phage lysis evolves as bet-hedging strategy that maximizes phage spread, regardless of whether colicin is present or not. We conclude that the Collb system, lacking its own lysis gene, is making use of the evolutionary stable phage strategy to be released. Prophage lysis genes are highly prevalent in nontyphoidal Salmonella genomes. This suggests that the release of Collb by temperate phages is widespread. In conclusion, our findings shed new light on the evolution and ecology of group B colicin systems.IMPORTANCE Bacteria are excellent model organisms to study mechanisms of social evolution. The production of public goods, e.g., toxin release by cell lysis in clonal bacterial populations, is a frequently studied example of cooperative behavior. Here, we analyze evolutionary stabilization of toxin release by the enteric pathogen Salmonella. The release of colicin lb (Collb), which is used by Salmonella to gain an edge against competing microbiota following infection, is coupled to bacterial lysis mediated by temperate phages. Here, we show that phage-dependent lysis and subsequent release of colicin and phage particles occurs only in part of the Collbexpressing Salmonella population. This phenotypic heterogeneity in lysis, which represents an essential step in the temperate phage life cycle, has evolved as a bethedging strategy under fluctuating environments such as the gastrointestinal tract. Our findings suggest that prophages can thereby evolutionarily stabilize costly toxin release in bacterial populations. AU - Spriewald, S.* AU - Stadler, E.* AU - Hense, B.A. AU - Münch, P.C.* AU - McHardy, A.C.* AU - Weiss, A.S.* AU - Obeng, N.* AU - Müller, J. AU - Stecher, B.* C1 - 59724 C2 - 48996 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Evolutionary stabilization of cooperative toxin production through a bacterium-plasmid-phage interplay. JO - mBio VL - 11 IS - 4 PB - Amer Soc Microbiology PY - 2020 SN - 2150-7511 ER - TY - JOUR AB - Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery.IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such "antiviral" compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients. AU - Stubbe, M.* AU - Mai, J.* AU - Paulus, C.* AU - Stubbe, H.C.* AU - Berscheminski, J.* AU - Karimi, M.* AU - Hofmann, S.* AU - Weber, E. AU - Hadian, K. AU - Hay, R.* AU - Groitl, P.* AU - Nevels, M.* AU - Dobner, T.* AU - Schreiner, S. C1 - 58624 C2 - 48513 TI - Viral DNA binding protein SUMOylation promotes PML nuclear body localization next to viral replication centers. JO - mBio VL - 11 IS - 2 PY - 2020 SN - 2150-7511 ER - TY - JOUR AB - It is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available for Neurospora crassa to investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCE In fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity. AU - Hassan, L.* AU - Lin, L.* AU - Sorek, H.* AU - Sperl, L.E. AU - Goudoulas, T.* AU - Hagn, F. AU - Germann, N.* AU - Tian, C.* AU - Benz, J.P.* C1 - 56558 C2 - 47136 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Cross-talk of cellulose and mannan perception pathways leads to inhibition of cellulase production in several filamentous fungi. JO - mBio VL - 10 IS - 4 PB - Amer Soc Microbiology PY - 2019 SN - 2150-7511 ER - TY - JOUR AB - The human persistent and oncogenic Epstein-Barr virus (EBV) was one of the first viruses that were described to express viral microRNAs (miRNAs). These have been proposed to modulate many host and viral functions, but their predominant role in vivo has remained unclear. We compared recombinant EBVs expressing or lacking miRNAs during in vivo infection of mice with reconstituted human immune system components and found that miRNA-deficient EBV replicates to lower viral titers with decreased frequencies of proliferating EBV-infected B cells. In response, activated cytotoxic EBV-specific T cells expand to lower frequencies than during infection with miRNA-expressing EBV. However, when we depleted CD8(+) T cells the miRNA-deficient virus reached similar viral loads as wild-type EBV, increasing by more than 200-fold in the spleens of infected animals. Furthermore, CD8(+) T cell depletion resulted in lymphoma formation in the majority of animals after miRNA-deficient EBV infection, while no tumors emerged when CD8(+) T cells were present. Thus, miRNAs mainly serve the purpose of immune evasion from T cells in vivo and could become a therapeutic target to render EBV-associated malignancies more immunogenic.IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the human population and usually persists asymptomatically within its host. Nevertheless, EBV is the causative agent for infectious mononucleosis (IM) and for lymphoproliferative disorders, including Burkitt and Hodgkin lymphomas. The immune system of the infected host is thought to prevent tumor formation in healthy virus carriers. EBV was one of the first viruses described to express miRNAs, and many host and viral targets were identified for these in vitro. However, their role during EBV infection in vivo remained unclear. This work is the first to describe that EBV miRNAs mainly increase viremia and virus-associated lymphomas through dampening antigen recognition by adaptive immune responses in mice with reconstituted immune responses. Currently, there is no prophylactic or therapeutic treatment to restrict IM or EBV-associated malignancies; thus, targeting EBV miRNAs could promote immune responses and limit EBV-associated pathologies. AU - Murer, A.* AU - Rühl, J.* AU - Zbinden, A.* AU - Capaul, R.* AU - Hammerschmidt, W. AU - Chijioke, O.* AU - Münz, C.* C1 - 55203 C2 - 46237 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - MicroRNAs of Epstein-Barr Virus attenuate T-cell-mediated immune control in vivo. JO - mBio VL - 10 IS - 1 PB - Amer Soc Microbiology PY - 2019 SN - 2150-7511 ER - TY - JOUR AU - Murer, A.* AU - Rühl, J.* AU - Zbinden, A.* AU - Capaul, R.* AU - Hammerschmidt, W. AU - Chijioke, O.* AU - Münz, C.* C1 - 56621 C2 - 47210 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - MicroRNAs of Epstein-Barr Virus attenuate T-cell-mediated immune control in vivo (vol 10, e01941-18, 2019). JO - mBio VL - 10 IS - 4 PB - Amer Soc Microbiology PY - 2019 SN - 2150-7511 ER - TY - JOUR AB - Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells.IMPORTANCE The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV's multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV's prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established. AU - Pich, D. AU - Mrozek-Gorska, P. AU - Bouvet, M. AU - Sugimoto, A. AU - Akidil, E. AU - Grundhoff, A.* AU - Hamperl, S. AU - Ling, P.D.* AU - Hammerschmidt, W. C1 - 56956 C2 - 47442 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - First days in the life of naïve human B-lymphocytes infected with Epstein-Barr Virus. JO - mBio VL - 10 IS - 5 PB - Amer Soc Microbiology PY - 2019 SN - 2150-7511 ER - TY - JOUR AB - Diet can influence the composition of the human microbiome, and yet relatively few dietary ingredients have been systematically investigated with respect to their impact on the functional potential of the microbiome. Dietary resistant starch (RS) has been shown to have health benefits, but we lack a mechanistic understanding of the metabolic processes that occur in the gut during digestion of RS. Here, we collected samples during a dietary crossover study with diets containing large or small amounts of RS. We determined the impact of RS on the gut microbiome and metabolic pathways in the gut, using a combination of "omics" approaches, including 16S rRNA gene sequencing, metaproteomics, and metabolomics. This multiomics approach captured changes in the abundance of specific bacterial species, proteins, and metabolites after a diet high in resistant starch (HRS), providing key insights into the influence of dietary interventions on the gut microbiome. The combined data showed that a high-RS diet caused an increase in the ratio of Firmicutes to Bacteroidetes, including increases in relative abundances of some specific members of the Firmicutes and concurrent increases in enzymatic pathways and metabolites involved in lipid metabolism in the gut.IMPORTANCE This work was undertaken to obtain a mechanistic understanding of the complex interplay between diet and the microorganisms residing in the intestine. Although it is known that gut microbes play a key role in digestion of the food that we consume, the specific contributions of different microorganisms are not well understood. In addition, the metabolic pathways and resultant products of metabolism during digestion are highly complex. To address these knowledge gaps, we used a combination of molecular approaches to determine the identities of the microorganisms in the gut during digestion of dietary starch as well as the metabolic pathways that they carry out. Together, these data provide a more complete picture of the function of the gut microbiome in digestion, including links between an RS diet and lipid metabolism and novel linkages between specific gut microbes and their metabolites and proteins produced in the gut. AU - Maier, T.V. AU - Lucio, M. AU - Lee, L.H.* AU - VerBerkmoes, N.C.* AU - Brislawn, C.J.* AU - Bernhardt, J.* AU - Lamendella, R.* AU - McDermott, J.E.* AU - Bergeron, N.* AU - Heinzmann, S.S. AU - Morton, J.T.* AU - González, A.J.* AU - Ackermann, G.* AU - Knight, R.* AU - Riedel, K.* AU - Krauss, R.M.* AU - Schmitt-Kopplin, P. AU - Jansson, J.K.* C1 - 52156 C2 - 43782 CY - Washington TI - Impact of dietary resistant starch on the human gut microbiome, metaproteome, and metabolome. JO - mBio VL - 8 IS - 5 PB - Amer Soc Microbiology PY - 2017 SN - 2150-7511 ER - TY - JOUR AB - Signal peptides are a cornerstone mechanism for cellular protein localization, yet until now experimental determination of signal peptides has come from only a narrow taxonomic sampling. As a result, the dominant view is that Sec-cleaved signal peptides in prokaryotes are defined by a canonical AxA motif. Although other residues are permitted in the motif, alanine is by far the most common. Here we broadly examine proteomics data to reveal the signal peptide sequences for 32 bacterial and archaeal organisms from nine phyla and demonstrate that this alanine preference is not universal. Discoveries include fundamentally distinct signal peptide motifs from Alphaproteobacteria, Spirochaetes, Thermotogae and Euryarchaeota. In these novel motifs, alanine is no longer the dominant residue but has been replaced in a different way for each taxon. Surprisingly, divergent motifs correlate with a proteome-wide reduction in alanine. Computational analyses of similar to 1,500 genomes reveal numerous major evolutionary clades which have replaced the canonical signal peptide sequence with novel motifs. IMPORTANCE This article replaces a widely held general model with a more detailed model describing phylogenetically correlated variation in motifs for Sec secretion. AU - Payne, S.H.* AU - Bonissone, S.* AU - Wu, S.* AU - Brown, R.N.* AU - Ivankov, D.N.* AU - Frishman, D. AU - Pasa-Tolic, L.* AU - Smith, R.D.* AU - Pevzner, P.A.* C1 - 22422 C2 - 30898 TI - Unexpected diversity of signal peptides in prokaryotes. JO - mBio VL - 3 IS - 6 PB - American Society of Microbiology PY - 2012 SN - 2150-7511 ER -