The autophagy machinery interacts with EBV capsids during viral envelope release.

Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.

EBNA2-EBF1 complexes promote MYC expression and metabolic processes driving S-phase progression of Epstein-Barr virus-infected B cells.

Epstein-Barr virus (EBV) is a human tumor virus which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen 2 (EBNA2) is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA-binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor 1 (EBF1), a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. The α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F, as well as multiple metabolic processes linked to cell cycle progression are impaired in EBVΔα1-infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV-infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1.

In infected cells, Epstein-Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA's transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs.

MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection.

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.

CD27 is required for protective lytic EBV antigen-specific CD8+ T-cell expansion.

Primary immunodeficiencies in the costimulatory molecule CD27 and its ligand, CD70, predispose for pathologies of uncontrolled Epstein-Barr virus (EBV) infection in nearly all affected patients. We demonstrate that both depletion of CD27+ cells and antibody blocking of CD27 interaction with CD70 cause uncontrolled EBV infection in mice with reconstituted human immune system components. While overall CD8+ T-cell expansion and composition are unaltered after antibody blocking of CD27, only some EBV-specific CD8+ T-cell responses, exemplified by early lytic EBV antigen BMLF1-specific CD8+ T cells, are inhibited in their proliferation and killing of EBV-transformed B cells. This suggests that CD27 is not required for all CD8+ T-cell expansions and cytotoxicity but is required for a subset of CD8+ T-cell responses that protect us from EBV pathology.

PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice.

While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): a novel function for the viral EBNA-LP protein.

Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides-besides transcription-an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP-together with the RBM4 splicing factor-in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

Epstein-Barr virus reprograms human B lymphocytes immediately in the prelatent phase of infection.

Epstein-Barr virus (EBV) is a human tumor virus and a model of herpesviral latency. The virus efficiently infects resting human B lymphocytes and induces their continuous proliferation in vitro, which mimics certain aspects of EBV's oncogenic potential in vivo. How lymphoblastoid cell lines (LCLs) evolve from the infected lymphocytes is uncertain. We conducted a systematic time-resolved longitudinal study of cellular functions and transcriptional profiles of newly infected naïve primary B lymphocytes. EBV reprograms the cells comprehensively and globally. Rapid and extensive transcriptional changes occur within 24 h and precede any metabolic and phenotypic changes. Within 72 h, the virus activates the cells, changes their phenotypes with respect to cell size, RNA, and protein content, and induces metabolic pathways to cope with the increased demand for energy, supporting an efficient cell cycle entry on day 3 postinfection. The transcriptional program that EBV initiates consists of 3 waves of clearly discernable clusters of cellular genes that peak on day 2, 3, or 4 and regulate RNA synthesis, metabolic pathways, and cell division, respectively. Upon onset of cell doublings on day 4, the cellular transcriptome appears to be completely reprogrammed to support the proliferating cells, but 3 additional clusters of EBV-regulated genes fine-tune cell signaling, migration, and immune response pathways, eventually. Our study reveals that more than 11,000 genes are regulated upon EBV infection as naïve B cells exit quiescence to enter a germinal center-like differentiation program, which culminates in immortalized, proliferating cells that partially resemble plasmablasts and early plasma cells.

B cells latently infected with murine gammaherpesvirus 68 (MHV-68) are present in the mouse thymus-A step toward immune evasion?

We show that latently gammaherpesvirus-infected B cells are present in the thymus. This could result in a functional T-cell tolerance against certain viral epitopes. It is conceivable that also antigens from other viruses or pathogens may be conveyed to the thymus for their immune evasion.

Spatiotemporally Skewed Activation of Programmed Cell Death Receptor 1-Positive T Cells after Epstein-Barr Virus Infection and Tumor Development in Long-Term Fully Humanized Mice.

Humanized mice developing functional human T cells endogenously and capable of recognizing cognate human leukocyte antigen-matched tumors are emerging as relevant models for studying human immuno-oncology in vivo. Herein, mice transplanted with human CD34+ stem cells and bearing endogenously developed human T cells for >15 weeks were infected with an oncogenic recombinant Epstein-Barr virus (EBV), encoding enhanced firefly luciferase and green fluorescent protein. EBV-firefly luciferase was detectable 1 week after infection by noninvasive optical imaging in the spleen, from where it spread rapidly and systemically. EBV infection resulted into a pronounced immunologic skewing regarding the expansion of CD8+ T cells in the blood outnumbering the CD4+ T and CD19+ B cells. Furthermore, within 10 weeks of infections, mice developing EBV-induced tumors had significantly higher absolute numbers of CD8+ T cells in lymphatic tissues than mice controlling tumor development. Tumor outgrowth was paralleled by an up-regulation of the programmed cell death receptor 1 on CD8+ and CD4+ T cells, indicative for T-cell dysfunction. Histopathological examinations and in situ hybridizations for EBV in tumors, spleen, liver, and kidney revealed foci of EBV-infected cells in perivascular regions in close association with programmed cell death receptor 1-positive infiltrating lymphocytes. The strong spatiotemporal correlation between tumor development and the T-cell dysfunctional status seen in this viral oncogenesis humanized model replicates observations obtained in the clinical setting.

Epstein-Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells.

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4+ T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8+ T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8+ T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8+ T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8+ T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4+ but also by antiviral CD8+ T cells.

MicroRNAs of Epstein-Barr Virus Control Innate and Adaptive Antiviral Immunity.

Epstein-Barr virus (EBV) has established lifelong infection in more than 90% of humanity. While infection is usually controlled by the immune system, the human host fails to completely eliminate the pathogen. Several herpesviral proteins are known to act as immunoevasins, preventing or reducing recognition of EBV-infected cells. Only recently were microRNAs of EBV identified to reduce immune recognition further. This Gem summarizes what we know about immunomodulatory microRNAs of herpesviruses.

K1 and K15 of Kaposi's Sarcoma-Associated Herpesvirus Are Partial Functional Homologues of Latent Membrane Protein 2A of Epstein-Barr Virus.

UNLABELLED: The human herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are associated with Hodgkin's lymphoma (HL) and Primary effusion lymphomas (PEL), respectively, which are B cell malignancies that originate from germinal center B cells. PEL cells but also a quarter of EBV-positive HL tumor cells do not express the genuine B cell receptor (BCR), a situation incompatible with survival of normal B cells. EBV encodes LMP2A, one of EBV's viral latent membrane proteins, which likely replaces the BCR's survival signaling in HL. Whether KSHV encodes a viral BCR mimic that contributes to oncogenesis is not known because an experimental model of KSHV-mediated B cell transformation is lacking. We addressed this uncertainty with mutant EBVs encoding the KSHV genes K1 or K15 in lieu of LMP2A and infected primary BCR-negative (BCR(-)) human B cells with them. We confirmed that the survival of BCR(-) B cells and their proliferation depended on an active LMP2A signal. Like LMP2A, the expression of K1 and K15 led to the survival of BCR(-) B cells prone to apoptosis, supported their proliferation, and regulated a similar set of cellular target genes. K1 and K15 encoded proteins appear to have noncomplementing, redundant functions in this model, but our findings suggest that both KSHV proteins can replace LMP2A's key activities contributing to the survival, activation and proliferation of BCR(-) PEL cells in vivo. IMPORTANCE: Several herpesviruses encode oncogenes that are receptor-like proteins. Often, they are constitutively active providing important functions to the latently infected cells. LMP2A of Epstein-Barr virus (EBV) is such a receptor that mimics an activated B cell receptor, BCR. K1 and K15, related receptors of Kaposi's sarcoma-associated herpesvirus (KSHV) expressed in virus-associated tumors, have less obvious functions. We found in infection experiments that both viral receptors of KSHV can replace LMP2A and deliver functions similar to the endogenous BCR. K1, K15, and LMP2A also control the expression of a related set of cellular genes in primary human B cells, the target cells of EBV and KSHV. The observed phenotypes, as well as the known characteristics of these genes, argue for their contributions to cellular survival, B cell activation, and proliferation. Our findings provide one possible explanation for the tumorigenicity of KSHV, which poses a severe problem in immunocompromised patients.

The Epigenetic Life Cycle of Epstein-Barr Virus.

Ever since the discovery of Epstein-Barr virus (EBV) more than 50 years ago, this virus has been studied for its capacity to readily establish a latent infection, which is the prominent hallmark of this member of the herpesvirus family. EBV has become an important model for many aspects of herpesviral latency, but the molecular steps and mechanisms that lead to and promote viral latency have only emerged recently. It now appears that the virus exploits diverse facets of epigenetic gene regulation in the cellular host to establish a latent infection. Most viral genes are transcriptionally repressed, and viral chromatin is densely compacted during EBV's latent phase, but latent infection is not a dead end. In order to escape from this phase, epigenetic silencing must be reverted efficiently and quickly. It appears that EBV has perfected a clever strategy to overcome transcriptional repression of its many lytic genes to initiate virus de novo synthesis within a few hours after induction of its lytic cycle. This review tries to summarize the known molecular mechanisms, the current models, concepts, and ideas underlying this viral strategy. This review also attempts to identify and address gaps in our current understanding of EBV's epigenetic mechanisms within the infected cellular host.

c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs.

CpG methylation in mammalian DNA is known to interfere with gene expression by inhibiting the binding of transactivators to their cognate sequence motifs or recruiting proteins involved in gene repression. An Epstein-Barr virus-encoded transcription factor, Zta, was the first example of a sequence-specific transcription factor that preferentially recognizes and selectively binds DNA sequence motifs with methylated CpG residues, reverses epigenetic silencing and activates gene transcription. The DNA binding domain of Zta is homologous to c-Fos, a member of the cellular AP-1 (activator protein 1) transcription factor family, which regulates cell proliferation and survival, apoptosis, transformation and oncogenesis. We have identified a novel AP-1 binding site termed meAP-1, which contains a CpG dinucleotide. If methylated, meAP-1 sites are preferentially bound by the AP-1 heterodimer c-Jun/c-Fos in vitro and in cellular chromatin in vivo. In activated human primary B cells, c-Jun/c-Fos locates to these methylated elements in promoter regions of transcriptionally activated genes. Reminiscent of the viral Zta protein, c-Jun/c-Fos is the first identified cellular member of the AP-1 family of transactivators that can induce expression of genes with methylated, hence repressed promoters, reversing epigenetic silencing.

RNAs in Epstein-Barr virions control early steps of infection.

Herpesviruses are dsDNA viruses, but their virions may additionally contain RNAs that can be transduced to recipient cells. The biological functions of herpes virion RNA species are unknown. Here we address this issue for EBV, a widespread human herpesvirus with oncogenic potential. We show that EBV-derived particles that include virions, virus-like particles, and subviral vesicles contain viral mRNAs, microRNAs, and other noncoding RNAs. Viral RNAs were transduced during infection and deployed immediate functions that enhanced EBV's capacity to transform primary B cells. Among these transduced viral RNAs, BZLF1 transcripts transactivated viral promoters triggering the prelatent phase of EBV infection, noncoding EBV-encoded RNA transcripts induced cellular cytokine synthesis, and BNLF2a mRNA led to immune evasion that prevented T-cell responses to newly infected B cells. Hence, transduced viral RNAs govern critical processes immediately after infection of B cells with EBV and likely play important roles in herpesviral infection in general.

BZLF1 governs CpG-methylated chromatin of Epstein-Barr Virus reversing epigenetic repression.

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.

The EBV immunoevasins vIL-10 and BNLF2a protect newly infected B cells from immune recognition and elimination.

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.