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Introduction N6‐methyladenosine (m6A) is the most prevalent modification that occurs in messenger RNA (mRNA), affecting mRNA splicing, translation, and stability. This modification is reversible, and its related biological functions are mediated by “writers,” “erasers,” and “readers.” The field of viral epitranscriptomics and the role of m6A modification in virus–host interaction have attracted much attention recently. When Epstein–Barr virus (EBV) infects a human B lymphocyte, it goes through three phases: the pre‐latent phase, latent phase, and lytic phase. Little is known about the viral and cellular m6A epitranscriptomes in EBV infection, especially in the pre‐latent phase during de novo infection. Methods Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) and MeRIP‐RT‐qPCR were used to determine the m6A‐modified transcripts during de novo EBV infection. RIP assay was used to confirm the binding of EBNA2 and m6A readers. Quantitative reverse‐transcription polymerase chain reaction (RT‐qPCR) and Western blot analysis were performed to test the effect of m6A on the host and viral gene expression. Results Here, we provided mechanistic insights by examining the viral and cellular m6A epitranscriptomes during de novo EBV infection, which is in the pre‐latent phase. EBV EBNA2 and BHRF1 were highly m6A‐modified upon EBV infection. Knockdown of METTL3 (a “writer”) decreased EBNA2 expression levels. The emergent m6A modifications induced by EBV infection preferentially distributed in 3ʹ untranslated regions of cellular transcripts, while the lost m6A modifications induced by EBV infection preferentially distributed in coding sequence regions of mRNAs. EBV infection could influence the host cellular m6A epitranscriptome. Conclusions These results reveal the critical role of m6A modification in the process of de novo EBV infection., The viral and cellular N6‐methyladenosine (m6A) epitranscriptomes were changed during de novo Epstein–Barr (EBV) infection in B cells. EBV EBNA2 was a significant m6A‐modified viral transcript, and cellular TLR9 and FAS genes' m6A modification levels are also modulated by EBV infection.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases including cancers of lymphoid and epithelial cells. EBV-cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase is essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor Tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation post-reactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress, and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication such as chronic active EBV infection and post-transplant lymphoproliferative disorders.IMPORTANCEProduction of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans but in so doing, can set the stage for development of EBV-cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus, and by interfering with Tsg101, prevent EBV release from lytically-active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely-used prazoles as candidates to prevent development of post-transplant EBV-lymphomas.

Significance Multiple families of DNA viruses including herpesviruses amplify their genomes in nuclear sites termed replication compartments. What benefits the viruses gain by this spatial and temporal control is unclear. We have analyzed the replication compartments induced by Epstein–Barr virus (EBV) and its DNA amplification in detail to elucidate their functions and regulation in EBV’s productive cycle. We found that EBV uses its replication compartments to coordinate the amplification of its genomes: Each compartment is seeded by single viral DNAs, each compartment supports similar levels of viral DNA synthesis, and each completes this synthesis as the replication machinery declines within it. Thus, replication compartments not only exclude cellular DNA synthesis but are hubs for the coordination of viral DNA amplification., Herpesviruses must amplify their DNA to load viral particles and they do so in replication compartments. The development and functions of replication compartments during DNA amplification are poorly understood, though. Here we examine 2 functionally distinct replicons in the same cells to dissect DNA amplification within replication compartments. Using a combination of single-cell assays, computational modeling, and population approaches, we show that compartments initially were seeded by single genomes of Epstein–Barr virus (EBV). Their amplification subsequently took 13 to 14 h in individual cells during which their compartments occupied up to 30% of the nucleus and the nuclear volume grew by 50%. The compartmental volumes increased in proportion to the amount of DNA and viral replication proteins they contained. Each compartment synthesized similar levels of DNA, indicating that the total number of compartments determined the total levels of DNA amplification. Further, the amplification, which depended on the number of origins, was regulated differently early and late during the lytic phase; early during the lytic phase, the templates limited DNA synthesis, while later the templates were in excess, coinciding with a decline in levels of the viral replication protein, BMRF1, in the replication compartments. These findings show that replication compartments are factories in which EBV DNA amplification is both clonal and coordinated.

The success of long-term host–virus partnerships is predicated on the ability of the host to limit the destructive potential of the virus and the virus’s skill in manipulating its host to persist undetected yet replicate efficiently when needed. By mastering such skills, herpesviruses persist silently in their hosts, though perturbations in this host–virus equilibrium can result in disease. The heterochromatin machinery that tightly regulates endogenous retroviral elements and pericentromeric repeats also silences invading genomes of alpha-, beta-, and gammaherpesviruses. That said, how these viruses disrupt this constitutive heterochromatin machinery to replicate and spread, particularly in response to disparate lytic triggers, is unclear. Here, we review how the cancer-causing gammaherpesvirus Epstein–Barr virus (EBV) uses the inflammasome as a security system to alert itself of threats to its cellular home as well as to flip the virus-encoded lytic switch, allowing it to replicate and escape in response to a variety of lytic triggers. EBV provides the first example of an infectious agent able to actively exploit the inflammasome to spark its replication. Revealing an unexpected link between the inflammasome and the epigenome, this further brings insights into how the heterochromatin machinery uses differential strategies to maintain the integrity of the cellular genome whilst guarding against invading pathogens. These recent insights into EBV biology and host–viral epigenetic regulation ultimately point to the NLRP3 inflammasome as an attractive target to thwart herpesvirus reactivation.

Antigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication. IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

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 (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal-cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that, through the activities of the viral protein kinase that is conserved across herpesviruses and two cellular proteins ATM and KAP1, a lytic cycle amplification loop is established - and that disruption of this loop disables the EBV lytic cascade. We therefore devised a high throughput screening assay, screened a small molecule compound library, and identified seventeen candidates that impair the release of lytically-replicated EBV. The identified compounds will i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses and ii) be developed into anti-cancer agents as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for anti-herpesviral and anti-cancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even anti-herpesviral potential as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both anti-herpesviral and anti-cancer drugs.

Epstein-Barr virus (EBV) is a model of herpesvirus latency and epigenetic changes. The virus preferentially infects human B-lymphocytes (and also other cell types) but does not turn them straight into virus factories. Instead, it establishes a strictly latent infection in them and concomitantly induces the activation and proliferation of infected B cells. How the virus establishes latency in its target cells is only partially understood, but its latent state has been studied intensively by many. During latency, several copies of the viral genome are maintained as minichromosomes in the nucleus. In latently infected cells, most viral genes are epigenetically repressed by cellular chromatin constituents and DNA methylation, but certain EBV genes are spared and remain expressed to support the latent state of the virus in its host cell. Latency is not a dead end, but the virus can escape from this state and reactivate. Reactivation is a coordinated process that requires the removal of repressive chromatin components and a gain in accessibility for viral and cellular factors and machines to support the entire transcriptional program of EBV’s ensuing lytic phase. We have a detailed picture of the initiating events of EBV’s lytic phase, which are orchestrated by a single viral protein – BZLF1. Its induced expression can lead to the expression of all lytic viral proteins, but initially it fosters the non-licensed amplification of viral DNA that is incorporated into preformed capsids. In the virions, the viral DNA is free of histones and lacks methylated cytosine residues which are lost during lytic DNA amplification. This review provides an overview of EBV’s dynamic epigenetic changes, which are an integral part of its ingenious lifestyle in human host cells.

Viruses activate inflammasomes but then subvert resulting inflammatory responses to avoid elimination. We asked whether viruses could instead use such activated or primed inflammasomes to directly aid their propagation and spread. Since herpesviruses are experts at coopting cellular functions, we investigated whether Epstein−Barr virus (EBV), an oncoherpesvirus, exploits inflammasomes to activate its replicative or lytic phase. Indeed, our experiments reveal that EBV exploits several inflammasome sensors to actually activate its replicative phase from quiescence/latency. In particular, TXNIP, a key inflammasome intermediary, causes assembly of the NLRP3 inflammasome, resulting in caspase-1−mediated depletion of the heterochromatin-inducing epigenetic repressor KAP1/TRIM28 in a subpopulation of cells. As a result, only TXNIP(hi)KAP1(lo) cells, that is, in a primed/prolytic state, turn expression of the replication/lytic/reactivation switch protein on to enter the replicative phase. Our findings 1) demonstrate that EBV dovetails its escape strategy to a key cellular danger-sensing mechanism, 2) indicate that transcription may be regulated by KAP1 abundance aside from canonical regulation through its posttranslational modification, 3) mechanistically link diabetes, which frequently activates the NLRP3 inflammasome, to deregulation of a tumor virus, and 4) demonstrate that B lymphocytes from NOMID (neonatal onset multisystem inflammatory disease) patients who have NLRP3 mutations and suffer from hyperactive innate responses are defective in controlling a herpesvirus.

NK cells have been reported to respond against EBV-infected B cells in the lytic cycle and to control the viral infection involving IFN-γ secretion. Early reports proposed a role for NK cell Ab-dependent cellular cytotoxicity (ADCC) triggered via FcγR-IIIA (CD16) in the response to EBV. In the current study, we revisited this issue, showing that serum from EBV+ individuals triggered vigorous NK cell degranulation and cytokine production (i.e., TNF-α and IFN-γ) against EBV-infected cells, enhancing NK cell activation. The effect was preferentially directed against cells in the lytic phase and was associated with surface expression of the gp350/220 envelope Ag. In contrast, binding of gp350+ particles, released by EBV-infected cells, to B cell lines or autologous primary B lymphocytes also promoted specific Ab-dependent NK cell degranulation and TNF-α production but induced minimal IFN-γ secretion. In that case, target cell damage appeared marginal compared with the effect of a control anti-CD20 Ab (rituximab) at concentrations that triggered similar NK cell activation, indicating that cell-associated gp350+ particles may divert the cytolytic machinery, impairing its direct action on the plasma membrane. These observations support that Ab-dependent NK cell activation plays an important role in the control of EBV, enhancing NK cell effector functions against infected B cells in the lytic cycle. In contrast, the data reveal that gp350+ particles bound to bystander B cells trigger Ab-dependent NK cell degranulation and TNF-α but not cytotoxicity or IFN-γ production, potentially favoring the progression of viral infection.

Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

Herpesviruses are ubiquitous, and infection by some, like Epstein-Barr virus (EBV), is nearly universal. To persist, EBV must periodically switch from a latent to a replicative/lytic phase. This productive phase is responsible for most herpesvirus-associated diseases. EBV encodes a latency-to-lytic switch protein which, upon activation, sets off a vectorially constrained cascade of gene expression that results in production of infectious virus. While triggering expression of the switch protein ZEBRA is essential to lytic cycle entry, sustaining its expression is equally important to avoid premature termination of the lytic cascade. We report that the viral protein kinase (vPK), encoded by a gene that is kinetically downstream of the lytic switch, sustains expression of ZEBRA, amplifies the lytic cascade, increasing virus production, and, importantly, prevents the abortive lytic cycle. We find that vPK, through a noncanonical site phosphorylation, activates the cellular phosphatidylinositol 3-kinase-related kinase ATM to cause phosphorylation of the heterochromatin enforcer KAP1/TRIM28 even in the absence of EBV genomes or other EBV proteins. Phosphorylation of KAP1 renders it unable to restrain ZEBRA, thereby further derepressing and sustaining its expression to culminate in virus production. This partnership with a host kinase and a transcriptional corepressor enables retrograde regulation by vPK of ZEBRA, an observation that is counter to the unidirectional regulation of gene expression reminiscent of most DNA viruses. IMPORTANCE Herpesviruses infect nearly all humans and persist quiescently for the life of the host. These viruses intermittently activate into the lytic phase to produce infectious virus, thereby causing disease. To ensure that lytic activation is not prematurely terminated, expression of the virally encoded lytic switch protein needs to be sustained. In studying Epstein-Barr virus, one of the most prevalent human herpesviruses that also causes cancer, we have discovered that a viral kinase activated by the viral lytic switch protein partners with a cellular kinase to deactivate a silencer of the lytic switch protein, thereby providing a positive feedback loop to ensure successful completion of the viral productive phase. Our findings highlight key nodes of interaction between the host and virus that could be exploited to treat lytic phase-associated diseases by terminating the lytic phase or kill cancer cells harboring herpesviruses by accelerating the completion of the lytic cascade.

The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade. IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

Epstein−Barr virus (EBV), a member of the Herpesviridae family, maintains a lifelong latent infection in human B cells. Switching from the latent to the lytic phase of its lifecycle allows the virus to replicate and spread. The viral lytic cycle is induced in infected cultured cells by drugs such as sodium butyrate and azacytidine. Lytic reactivation can be inhibited by natural products and pharmaceuticals. The anticonvulsant drugs valproic acid and valpromide inhibit EBV in Burkitt lymphoma cells. Therefore, other drugs that treat neurological and psychological disorders were investigated for effects on EBV lytic reactivation. Clozapine, an atypical antipsychotic drug used to treat schizophrenia and bipolar disorder, was found to inhibit the reactivation of the EBV lytic cycle. Levels of the viral lytic genes BZLF1, BRLF1, and BMLF1 were decreased by treatment with clozapine in induced Burkitt lymphoma cells. The effects on viral gene expression were dependent on the dose of clozapine, yet cells were viable at an inhibitory concentration of clozapine. One metabolite of clozapine—desmethylclozapine—also inhibited EBV lytic reactivation, while another metabolite—clozapine-N-oxide—had no effect. These drugs may be used to study cellular pathways that control the viral lytic switch in order to develop treatments for diseases caused by EBV.

Incidental findings on PET CT studies are common. The distribution of Ga-DOTATATE is dependent on cell surface expression of somatostatin receptors, which may be pathologic or physiologic. Osteoporosis circumscripta is the early lytic phase of Paget disease associated with well-defined osteopenia, most commonly seen within the skull on imaging. The appearance has been well demonstrated on Tc HDP/MDP bone scans. Here, we present the case of a 76-year-old man with a small bowel carcinoid tumor who underwent staging imaging with Ga-DOTATATE PET CT with the incidental finding of osteoporosis circumscripta.

The Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication in cis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

Epstein-Barr virus (EBV) is a ubiquitous human virus which infects almost all humans during their lifetime and following the acute phase, persists for the remainder of the life of the individual. EBV infects B lymphocytes leading to their immortalisation, with persistence of the EBV genome as an episome. In the latent phase, EBV is prevented from reactivating through efficient cytotoxic cellular immunity. EBV reactivates (lytic phase) under conditions of psychological stress with consequent weakening of cellular immunity, and EBV reactivation has been shown to occur in a subset of individuals with each of a variety of cancers, autoimmune diseases, the autoimmune-like disease, chronic fatigue syndrome/myalgic encephalitis and under other circumstances such as being an inpatient in an intensive care unit. Chronic EBV reactivation is an important mechanism in the pathogenesis of many such diseases, yet is rarely tested for in immunocompetent individuals. This review summarises the pathogenesis of EBV infection, EBV reactivation and its role in disease, and methods which may be used to detect it. Known inhibitors of EBV reactivation and replication are discussed, including drugs licensed for treatment of other herpesviruses, licensed or experimental drugs for various other indications, compounds at an early stage of drug development and nutritional constituents such as vitamins and dietary supplements.

Various aspects of human cytomegalovirus (HCMV) pathogenesis, including its ability to replicate in specific cells and tissues and the mechanism(s) of horizontal transmission, are not well understood, predominantly because of the strict species specificity exhibited by HCMV. Murine CMV (MCMV), which contains numerous gene segments highly similar to those of HCMV, has been useful for modeling some aspects of CMV pathogenesis; however, it remains essential to build relevant human cell-based systems to investigate how the HCMV counterparts function. The salivary gland epithelium is a site of persistence for both human and murine cytomegaloviruses, and salivary secretions appear to play an important role in horizontal transmission. Therefore, it is important to understand how HCMV is replicating within the glandular epithelial cells so that it might be possible to therapeutically prevent transmission. In the present study, we describe the development of a salivary epithelial model derived from primary human “salispheres.” Initial infection of these primary salivary cells with HCMV occurs in a manner similar to that reported for established epithelial lines, in that gH/gL/UL128/UL130/UL131A (pentamer)-positive strains can infect and replicate, while laboratory-adapted pentamer-null strains do not. However, while HCMV enters the lytic phase and produces virus in salivary epithelial cells, it fails to exhibit robust spread throughout the culture and persists in a low percentage of salivary cells. The present study demonstrates the utility of these primary tissue-derived cells for studying HCMV replication in salivary epithelial cells in vitro . IMPORTANCE Human cytomegalovirus (HCMV) infects the majority of the world’s population, and although it typically establishes a quiescent infection with little to no disease in most individuals, the virus is responsible for a variety of devastating sequelae in immunocompromised adults and in developing fetuses. Therefore, identifying the viral properties essential for replication, spread, and horizontal transmission is an important area of medical science. Our studies use novel human salivary gland-derived cellular models to investigate the molecular details by which HCMV replicates in salivary epithelial cells and provide insight into the mechanisms by which the virus persists in the salivary epithelium, where it gains access to fluids centrally important for horizontal transmission.

The development of nasopharyngeal carcinoma (NPC), a common cancer in Southeastern Asia, is closely associated with Epstein-Barr virus (EBV) infection; however, the aetiological role of EBV in NPC pathogenesis remains enigmatic. The life cycle of EBV in NPC patients is defined as latency II, while the antibodies specific to lytic phase proteins, as well as lytic genes, were highly expressed in NPC patients. The correlation between antibody levels and the progression of NPC has been reported in some studies; however, most of these studies focused on IgA antibodies, and the results in different articles were not consistent. In this study, we concurrently determined the levels of IgA and IgG antibodies specific to six purified recombinant EBV antigens associated with different replication statuses of EBV: EBNA1 associated with latency II; the non-structural antigens Zta, TK, EA-D and EA-R associated with immediate-early and early lytic phases; and the EBV matrix protein VCA p18, which is involved in late lytic phase. Levels of antibodies specific to immediate-early and early antigens were correlated with the tumour progression, especially tumour size. The levels of antibodies specific to some lytic phase antigens were also correlated with lymph node inclusion and metastasis. However, the antibody specific to the latency II antigen EBNA1 was not correlated with either tumour size or metastasis. Consistent with previous transcriptome studies, the results suggested both the expression of lytic phase genes at the protein level and the intermittent reactivation of EBV in NPC patients.

The oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.

The life cycle of EBV is regulated by epigenetic modifications, such as CpG methylation and histone modifications. Here, we found that the expression of EZH2, which encodes a histone H3K27 methyltransferase, was induced by EBV infection; therefore, we generated EZH2-KO cells to investigate the role of EZH2 in EBV-infected Akata B cells. Disruption of EZH2 resulted in increased expression of EBV genes during the lytic phase and, therefore, efficient viral replication and progeny production. Our results shed light on the mechanisms underlying reactivation from an epigenetic point of view and further suggest a role for EZH2 as a form of innate immunity that restricts viral replication in infected cells., Epigenetic modifications play a pivotal role in the expression of the genes of Epstein-Barr virus (EBV). We found that de novo EBV infection of primary B cells caused moderate induction of enhancer of zeste homolog 2 (EZH2), the major histone H3 lysine 27 (K27) methyltransferase. To investigate the role of EZH2, we knocked out the EZH2 gene in EBV-negative Akata cells by the CRISPR/Cas9 system and infected the cells with EBV, followed by selection of EBV-positive cells. During the latent state, growth of EZH2-knockout (KO) cells was significantly slower after infection compared to wild-type controls, despite similar levels of viral gene expression between cell lines. After induction of the lytic cycle by anti-IgG, KO of EZH2 caused notable induction of expression of both latent and lytic viral genes, as well as increases in both viral DNA replication and progeny production. These results demonstrate that EZH2 is crucial for the intricate epigenetic regulation of not only lytic but also latent gene expression in Akata cells. IMPORTANCE The life cycle of EBV is regulated by epigenetic modifications, such as CpG methylation and histone modifications. Here, we found that the expression of EZH2, which encodes a histone H3K27 methyltransferase, was induced by EBV infection; therefore, we generated EZH2-KO cells to investigate the role of EZH2 in EBV-infected Akata B cells. Disruption of EZH2 resulted in increased expression of EBV genes during the lytic phase and, therefore, efficient viral replication and progeny production. Our results shed light on the mechanisms underlying reactivation from an epigenetic point of view and further suggest a role for EZH2 as a form of innate immunity that restricts viral replication in infected cells.

Herpes-Simplex Virus 1 (HSV-1) infects most humans when they are young, sometimes with fatal consequences. Gene expression occurs in a temporal order upon lytic HSV-1 infection: immediate early (IE) genes are expressed, then early (E) genes, followed by late (L) genes. During this infection cycle, the HSV-1 genome has the potential for exposure to APOBEC3 (A3) proteins, a family of cytidine deaminases that cause C>U mutations on single-stranded DNA (ssDNA), often resulting in a C>T transition. We developed a computational model for the mutational pressure of A3 on the lytic cycle of HSV-1 to determine which viral kinetic gene class is most vulnerable to A3 mutations. Using in silico stochastic methods, we simulated the infectious cycle under varying intensities of A3 mutational pressure. We found that the IE and E genes are more vulnerable to A3 than L genes. We validated this model by analyzing the A3 evolutionary footprints in 25 HSV-1 isolates. We find that IE and E genes have evolved to underrepresent A3 hotspot motifs more so than L genes, consistent with greater selection pressure on IE and E genes. We extend this model to two-step infections, such as those of polyomavirus, and find that the same pattern holds for over 25 human Polyomavirus (HPyVs) genomes. Genes expressed earlier during infection are more vulnerable to mutations than those expressed later., Author summary The APOBEC family of cytidine deaminases are a component of innate immunity that can bind to viral DNA and cause mutations. We developed a computational model of the lytic life cycle of herpes simplex virus 1 (HSV-1), a large double-stranded DNA virus, to evaluate the potential effects of APOBEC on the virus and its evolution. The model considered three types of viral genes: Immediate Early (IE), Early (E) and Late (L). We found that genes expressed earliest in the lytic life cycle, E and particularly IE, are the most vulnerable to APOBEC-mediated mutations, because even low levels of mutation were sufficient to disrupt the infection process. We were able to validate the predictions of the computational model by analyzing published HSV-1 genomes, in particular by using software tools that quantify the extent to which each genome has evolved to reduce the number of DNA motifs (also known as “mutational hotspots”) that are preferred by APOBEC for mutation. Our analysis suggested that the IE and E genes have evolved to avoid APOBEC3 targeting. We extended our modeling and genomic analysis to human Polyomaviruses, which are smaller double-stranded DNA viruses with a simpler life cycle than HSV-1 and found similar results. These studies highlight the vulnerability to APOBEC of genes expressed early during viral infection and may suggest these genes as therapeutic targets.

Krüppel-associated box-domain zinc finger protein (KRAB-ZFP) transcriptional repressors recruit TRIM28/KAP1 to heterochromatinize the mammalian genome while also guarding the host by silencing invading foreign genomes. However, how a KRAB-ZFP recognizes target sequences in the natural context of its own or foreign genomes is unclear. Our studies on B-lymphocytes permanently harboring the cancer-causing Epstein-Barr virus (EBV) have shown that SZF1, a KRAB-ZFP, binds to several lytic/replicative phase genes to silence them, thereby promoting the latent/quiescent phase of the virus. As a result, unless SZF1 and its binding partners are displaced from target regions on the viral genome, EBV remains dormant, i.e. refractory to lytic phase-inducing triggers. As SZF1 also heterochromatinizes the cellular genome, we performed in situ footprint mapping on both viral and host genomes in physically separated B-lymphocytes bearing latent or replicative/active EBV genomes. By analyzing footprints, we learned that SZF1 recognizes the host genome through a repeat sequence-bearing motif near centromeres. Remarkably, SZF1 does not use this motif to recognize the EBV genome. Instead, it uses distinct binding sites that lack obvious similarities to each other or the above motif, to silence the viral genome. Virus mutagenesis studies show that these distinct binding sites are not only key to maintaining the established latent phase but also silencing the lytic phase in newly-infected cells, thus enabling the virus to establish latency and transform cells. Notably, these binding sites on the viral genome, when also present on the human genome, are not used by SZF1 to silence host genes during latency. This differential approach towards target site recognition may reflect a strategy by which the host silences and regulates genomes of persistent invaders without jeopardizing its own homeostasis., Author summary Heterochromatin marks silenced portions of the human genome. Heterochromatin also serves as a defense strategy to silence foreign genomes. Yet, how the heterochromatin inducing KRAB-ZFP-TRIM28 machinery recognizes target sites on the native genome, whether self or foreign, is unclear. Using Epstein-Barr virus-infected cells in which a KRAB-ZFP, SZF1, silences lytic/replicative-phase genes of the virus, we performed in situ mapping of ZFP-footprints on cell and viral genomes. We find that while the ZFP uses a repeat sequence-bearing motif to target pericentromeric regions, it uses non-consensus sites to target viral genes. These findings point towards i) a mechanism for directing constitutive heterochromatin and ii) a strategy that allows the host to use the same heterochromatin machinery to regulate an invader without deregulating itself.

The intrinsic properties of different viruses have driven their study. For example, the capacity for efficient productive infection of cultured cells by herpes simplex virus 1 has made it a paradigm for this mode of infection for herpesviruses in general. Epstein-Barr virus, another herpesvirus, has two properties that have driven its study: It causes human cancers, and it exhibits a tractable transition from its latent to its productive cycle in cell culture. Here, we review our understanding of the path Epstein-Barr virus follows to move from a latent infection to and through its productive cycle. We use information from human infections to provide a framework for describing studies in cell culture and, where possible, the molecular resolutions from these studies. We also pose questions whose answers we think are pivotal to understanding this path, and we provide answers where we can.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with three malignancies— Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Central to the pathogenesis of these diseases is the KSHV viral life cycle, which is composed of a quiescent latent phase and a replicative lytic phase. While the establishment of latency enables persistent KSHV infection and evasion of the host immune system, lytic replication is essential for the dissemination of the virus between hosts and within the host itself. The transition between these phases, known as lytic reactivation, is controlled by a complex set of environmental, host, and viral factors. The effects of these various factors converge on the regulation of two KSHV proteins whose functions facilitate each phase of the viral life cycle—latency-associated nuclear antigen (LANA) and the master switch of KSHV reactivation, replication and transcription activator (RTA). This review presents the current understanding of how the transition between the phases of the KSHV life cycle is regulated, how the various phases contribute to KSHV pathogenesis, and how the viral life cycle can be exploited as a therapeutic target.

Profound alterations in host cell nuclear architecture accompany the lytic phase of Epstein-Barr virus (EBV) infection. Viral replication compartments assemble, host chromatin marginalizes to the nuclear periphery, cytoplasmic poly(A)-binding protein translocates to the nucleus, and polyadenylated mRNAs are sequestered within the nucleus. Virus-induced changes to nuclear architecture that contribute to viral host shutoff (VHS) must accommodate selective processing and export of viral mRNAs. Here we describe additional previously unrecognized nuclear alterations during EBV lytic infection in which viral and cellular factors that function in pre-mRNA processing and mRNA export are redistributed. Early during lytic infection, before formation of viral replication compartments, two cellular pre-mRNA splicing factors, SC35 and SON, were dispersed from interchromatin granule clusters, and three mRNA export factors, Y14, ALY, and NXF1, were depleted from the nucleus. During late lytic infection, virus-induced nodular structures (VINORCs) formed at the periphery of viral replication compartments. VINORCs were composed of viral (BMLF1 and BGLF5) and cellular (SC35, SON, SRp20, and NXF1) proteins that mediate pre-mRNA processing and mRNA export. BHLF1 long noncoding RNA was invariably found in VINORCs. VINORCs did not contain other nodular nuclear cellular proteins (PML or coilin), nor did they contain viral proteins (BRLF1 or BMRF1) found exclusively within replication compartments. VINORCs are novel EBV-induced nuclear structures. We propose that EBV-induced dispersal and depletion of pre-mRNA processing and mRNA export factors during early lytic infection contribute to VHS; subsequent relocalization of these pre-mRNA processing and mRNA export proteins to VINORCs and viral replication compartments facilitates selective processing and export of viral mRNAs. IMPORTANCE In order to make protein, mRNA transcribed from DNA in the nucleus must enter the cytoplasm. Nuclear export of mRNA requires correct processing of mRNAs by enzymes that function in splicing and nuclear export. During the Epstein-Barr virus (EBV) lytic cycle, nuclear export of cellular mRNAs is blocked, yet export of viral mRNAs is facilitated. Here we report the dispersal and dramatic reorganization of cellular (SC35, SON, SRp20, Y14, ALY, and NXF1) and viral (BMLF1 and BGLF5) proteins that play key roles in pre-mRNA processing and export of mRNA. These virus-induced nuclear changes culminate in formation of VINORCs, novel nodular structures composed of viral and cellular RNA splicing and export factors. VINORCs localize to the periphery of viral replication compartments, where viral mRNAs reside. These EBV-induced changes in nuclear organization may contribute to blockade of nuclear export of host mRNA, while enabling selective processing and export of viral mRNA.

International audience; Epstein-Barr virus (EBV) is a widely distributed gamma-herpesvirus that has been associated with various cancers mainly from lymphocytic and epithelial origin. Although EBV-mediated oncogenesis has been associated with viral oncogenes expressed during latency, a growing set of evidence suggested that antiviral treatments directed against EBV lytic phase may contribute to prevent some forms of cancers, including EBV-positive Post-Transplant Lymphoproliferative Diseases. It is shown here that dipyridamole (DIP), a safe drug with favorable and broad pharmacological properties, inhibits EBV reactivation from B-cell lines. DIP repressed immediate early and early genes expression mostly through its ability to inhibit nucleoside uptake. Considering its wide clinical use, DIP repurposing could shortly be evaluated, alone or in combination with other antivirals, to treat EBV-related diseases where lytic replication plays a deleterious role.

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments. IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

Our previous study found that smoking was associated with an elevated level of the antibody against VCA in the Epstein-Barr virus (EBV) lytic phase, which was an important predictive marker of the risk of nasopharyngeal carcinoma (NPC). It remained unknown whether environmental factors were associated with the levels of other EBV antibodies, such as Zta-IgA, EA-IgA, EBNA1-IgA, and LMP1-IgA, in the lytic and latent infection periods. We aimed to investigate the possible environmental inducers that could affect EBV antibody levels in two independent healthy male populations from endemic NPC areas in South China (N = 1498) and non-endemic NPC areas in North China (N = 1961). We performed ELISA and immunoenzymatic assays to test the levels of antibodies specific to the EBV antigens. The seropositive rates of antibodies against the antigens expressed in both the EBV latent and lytic infection periods, namely, LMP1-IgA, EBNA1-IgA, and Zta-IgA, in endemic areas (28.65%, 5.43% and 14.49%, respectively) were significantly higher than those in non-endemic areas (14.43%, 1.07% and 6.32%, respectively). Smoking was associated with higher seropositivity for EBNA1-IgA (OR = 1.47, 95% CI = 1.12–1.93) and Zta-IgA (OR = 1.28, 95% CI = 0.99–1.66), with dose-response effects, while not associated with the levels of LMP1-IgA. In conclusion, smoking was an important environmental factor, which associated with increased levels of EBNA1-IgA, and Zta-IgA., Highlights • EBV antibodies against antigens were higher in healthy people in endemic areas than that in non-endemic areas of NPC. • Smoking was associated with higher seropositivity risk for EBNA1-IgA and Zta-IgA with dose-response relationships. • Smoking was not associated with seropositivity risk for LMP1-IgA. EBV, a class I oncovirus identified by WHO, is an important etiological factor of nasopharyngeal carcinoma (NPC). Numerous studies have demonstrated that higher levels of VCA-IgA, EBNA1-IgA, Zta-IgA against virus life cycle were good predict biomarkers for NPC risk. Our epidemiological study reported that several environmental factors, especially cigarette smoking, were associated the higher levels of Zta-IgA and EBNA1-IgA followed previously reported VCA-IgA against lytic period, the EBV reactivation marker; but not associated with LMP1-IgA, an antibody against a critical oncoprotein in latent period of EBV. It revealed that behavioral intervention, such as smoking cessation project, is very helpful in the primary prevention in endemic area.

The Epstein–Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, and occasionally switches from the latent state to the lytic cycle. In this report, we identified and examined the role of a lytic gene, BRRF2. We first prepared an antibody against BRRF2 and identified the gene product as a viral lytic protein expressed in B95-8 cells with late kinetics. Immunofluorescence revealed that BRRF2 localized in the cytoplasm of cells during the lytic phase. We also found that BRRF2 protein was phosphorylated in lytic cells, but the only viral protein kinase, BGLF4, was not involved in the phosphorylation. Knockout EBV and a repaired strain were then prepared, and we found that BRRF2 disruption did not affect viral gene expression and DNA replication, but decreased virus production. These results demonstrated that BRRF2 is involved in production of infectious progeny, although it is not essential for lytic replication.

A major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV + ) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV + cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers. IMPORTANCE Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV + cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies.

Kaposi’s sarcoma-associated herpesvirus (KSHV) primarily persists as a latent episome in infected cells. During latent infection, only a limited number of viral genes are expressed that help to maintain the viral episome and prevent lytic reactivation. The latent KSHV genome persists as a highly ordered chromatin structure with bivalent chromatin marks at the promoter-regulatory region of the major immediate-early gene promoter. Various stimuli can induce chromatin modifications to an active euchromatic epigenetic mark, leading to the expression of genes required for the transition from the latent to the lytic phase of KSHV life cycle. Enhanced replication and transcription activator (RTA) gene expression triggers a cascade of events, resulting in the modulation of various cellular pathways to support viral DNA synthesis. RTA also binds to the origin of lytic DNA replication to recruit viral, as well as cellular, proteins for the initiation of the lytic DNA replication of KSHV. In this review we will discuss some of the pivotal genetic and epigenetic factors that control KSHV reactivation from the transcriptionally restricted latent program.

Gammaherpesviruses (γHVs) have a dynamic strategy for lifelong persistence, involving productive infection, latency, and intermittent reactivation. In latency reservoirs, such as B lymphocytes, γHVs exist as viral episomes and express few viral genes. Although the ability of γHV to reactivate from latency and re-enter the lytic phase is challenging to investigate and control, it is known that the γHV replication and transcription activator (RTA) can promote lytic reactivation. In this study, we provide first evidence that RTA of murine γΗV68 (MHV68) selectively binds and enhances the activity of tyrosine-phosphorylated host STAT3. STAT3 is a transcription factor classically activated by specific tyrosine 705 phosphorylation (pTyr705-STAT3) in response to cytokine stimulation. pTyr705-STAT3 forms a dimer that avidly binds a consensus target site in the promoters of regulated genes, and our results indicate that RTA cooperatively enhances the ability of pTyr705-STAT3 to induce expression of a STAT3-responsive reporter gene. As indicated by coimmunoprecipitation, in latently infected B cells that are stimulated to reactivate MHV68, RTA bound specifically to endogenous pTyr705-STAT3. An in vitro binding assay confirmed that RTA selectively recognizes pTyr705-STAT3 and indicated that the C-terminal transactivation domain of RTA was required for enhancing STAT3-directed gene expression. The cooperation of these transcription factors may influence both viral and host genes. During MHV68 de novo infection, pTyr705-STAT3 promoted the temporal expression of ORF59, a viral replication protein. Our results demonstrate that MHV68 RTA specifically recognizes and recruits activated pTyr705-STAT3 during the lytic phase of infection.

The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ΔK3 ΔK5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K 5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K 3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1α-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP high and pK8.1-driven blue fluorescent protein-positive (BFP + ) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages. IMPORTANCE Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.

Kaposi's sarcoma-associated herpesvirus (KSHV) typically displays two different phases in its life cycle, the default latent phase and the lytic phase. There is a short period of lytic gene expression in the early stage of KSHV primary infection. The factors involved in the shutdown process of lytic gene expression are poorly identified. It has been shown that the latency-associated nuclear antigen (LANA) encoded by KSHV plays an important role in the establishment of viral latency. In screening, we identified a host protein, Krüppel-associated box domain-associated protein 1 (KAP1), that bound to LANA. We validated the interaction between LANA and KAP1 in vivo and in vitro , as well as their colocalization in the nucleus. We mapped out that LANA interacted with both the N- and C-terminal domains of KAP1. Based on the interface of LANA-KAP1 interaction determined, we proved that LANA recruited KAP1 to the RTA promoter region of the KSHV genome. We revealed that KAP1 was involved in transcriptional repression by LANA. We found multiple cooccupation sites of LANA and KAP1 on the whole KSHV genome by chromatin immunoprecipitation for sequencing (ChIP-seq) and demonstrated that LANA-recruited KAP1 played a critical role in the shutdown of lytic gene expression during the early stage of KSHV primary infection. Taken together, our data suggest that LANA interacts with KAP1 and represses lytic gene expression to facilitate the establishment of KSHV latency. IMPORTANCE Our study revealed the mechanism of transcriptional repression by LANA during KSHV primary infection, providing new insights into the process of KSHV latency establishment.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causatively linked to two B cell lymphoproliferative disorders, multicentric Castleman's disease and primary effusion lymphoma. Latently infected B cells are a major KSHV reservoir, and virus activation from tonsillar B cells can result in salivary shedding and virus transmission. Paradoxically, human B cells (primary and continuous) are notoriously refractory to infection, thus posing a major obstacle to the study of KSHV in this cell type. By performing a strategic search of human B cell lymphoma lines, we found that MC116 cells were efficiently infected by cell-free KSHV. Upon exposure to recombinant KSHV.219, enhanced green fluorescent protein reporter expression was detected in 17 to 20% of MC116 cells. Latent-phase transcription and protein synthesis were detected by reverse transcription-PCR and detection of latency-associated nuclear antigen expression, respectively, in cell lysates and individual cells. Selection based on the puromycin resistance gene in KSHV.219 yielded cultures with all cells infected. After repeated passaging of the selected KSHV-infected cells without puromycin, latent KSHV was maintained in a small fraction of cells. Infected MC116 cells could be induced into lytic phase with histone deacetylase inhibitors, as is known for latently infected non-B cell lines, and also selectively by the B cell-specific pathway involving B cell receptor cross-linking. Lytic-phase transition was documented by red fluorescent protein reporter expression, late structural glycoprotein (K8.1A, gH) detection, and infectious KSHV production. MC116 cells were CD27 − /CD10 + , characteristic of transitional B cells. These findings represent an important step in the establishment of an efficient continuous B cell line model to study the biologically relevant steps of KSHV infection. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes two serious pathologies of B cells, the antibody-producing cells of the immune system. B cells are a major reservoir for KSHV persistence in the body. Paradoxically, in the laboratory, B cells are extremely difficult to infect with KSHV; this problem greatly hinders scientific analysis of B cell infection. We describe our search for and successful identification of a stable human B cell line that can be efficiently infected by KSHV. Upon infection of these cells, the virus goes into a quiet latent phase, a characteristic feature of many herpesvirus infections. The virus can be triggered to enter an active lytic phase by treatments known to stimulate normal B cell functions. These findings suggest that the new B cell line will be a valuable model in which to study KSHV infection of this major target cell type.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV.

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., Author summary The life cycle of Kaposi Sarcoma herpesvirus involves both persistence in a latent form and productive replication to generate new viral particles. How the virus switches between latency and productive (‘lytic’) replication is only partially understood. Here we show that a viral homologue of interferon regulatory factors, vIRF2, antagonizes lytic protein expression in endothelial cells. It does this by inducing the expression of cellular interferon-regulated genes such as IFIT 1–3, which in turn dampens early viral gene expression. This observation suggests that vIRF2 allows KSHV to harness the interferon pathway to regulate early viral gene expression in endothelial cells.

Human cytomegalovirus (HCMV) lytic phase gene expression is repressed upon entry into myeloid lineage cells where the virus establishes latency. Lytic infection is not initiated because the tegument-delivered transactivator protein pp71 fails to enter the nucleus and inactivate the Daxx-mediated cellular intrinsic defense that silences the viral genome. When pp71 is expressed de novo in THP-1 monocytes, it localizes to the nucleus, inactivates the Daxx defense, and initiates lytic infection. We speculated that replacing the native viral promoter that drives pp71 expression with one that is highly and constitutively active in myeloid cells would permit pp71 de novo expression upon infection and that this newly expressed pp71 would accumulate in the nucleus, inactivate the intrinsic defense, and initiate the cascade of lytic gene expression. Surprisingly, we found that this promoter was still subject to normal silencing mechanisms in THP-1 monocytes and primary CD34 + cells, two independent myeloid lineage cells. A second constitutively active heterologous viral promoter located in a different region of the HCMV genome was also silenced in THP-1 and CD34 + cells. Furthermore, these two independent heterologous viral promoters inserted into three different regions of the HCMV genome in three different viral strains all required prior expression of the viral immediate early proteins for activation in fibroblasts. From this, we conclude that incorporation within the HCMV genome impacts the proclivity of heterologous viral promoters to initiate transcription. These observations have mechanistic implications for the expression of viral genes and transgenes during both lytic infection and latency.

Zinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity.

Reactivation of Epstein-Barr virus (EBV) from latency into the lytic phase of its life cycle allows the virus to spread among cells and between hosts. Valproic acid (VPA) inhibits initiation of the lytic cycle in EBV-infected B lymphoma cells. While VPA blocks viral lytic gene expression, it induces expression of many cellular genes, because it is a histone deacetylase (HDAC) inhibitor. Here we show, using derivatives of VPA, that blockade of EBV reactivation is separable from HDAC inhibition. Valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented expression of two EBV genes, BZLF1 and BRLF1, that mediate lytic reactivation. VPM also inhibited expression of a viral late gene, but not early genes, when BZLF1 was exogenously expressed. Unlike VPA, VPM did not activate lytic expression of Kaposi’s sarcoma-associated herpesvirus. Expression of cellular immediate-early genes, such as FOS and EGR1, is kinetically upstream of the EBV lytic cycle. VPM did not activate expression of these cellular immediate-early genes but decreased their level of expression when induced by butyrate, an HDAC inhibitor. VPM did not alter expression of several other cellular immediate-early genes, including STAT3, which were induced by the HDAC inhibitors in cells refractory to lytic induction. Therefore, VPM selectively inhibits both viral and cellular gene expression. VPA and VPM represent a new class of antiviral agents. The mechanism by which VPA and VPM block EBV reactivation may be related to their anticonvulsant activity., IMPORTANCE Epstein-Barr virus, (EBV), a human tumor virus, establishes a life-long latent infection. Reactivation of EBV into the lytic phase of its life cycle allows the virus to spread. Previously, we showed that EBV reactivation was blocked by valproic acid (VPA), an inhibitor of cellular histone deacetylases (HDACs). VPA alters the expression of thousands of cellular genes. In this study, we demonstrate that valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented initiation of the EBV lytic cycle. VPA induced lytic reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV), but VPM did not. Unlike VPA, VPM did not activate cellular immediate-early gene expression. VPM is a new type of antiviral agent. VPM will be useful in probing the mechanism of EBV lytic reactivation and may have therapeutic application.

Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein–Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2′-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation.

Amongst the pathologies associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV), multicentric Castleman's disease is distinctive for involvement of the lytic phase of the virus replication cycle. This B cell lymphoproliferative disorder has shown clinical responsiveness not only to generalized immunotherapy and cytotoxic chemotherapy, but also to inhibitors of herpesvirus DNA replication, consistent with the involvement of lytic phase of replication. These findings suggest that selective killing of virus-producing cells might represent a novel therapeutic strategy. We designed an immunotoxin, YC15-PE38, containing a single chain variable region fragment of a monoclonal antibody against KSHV glycoprotein H (gH) linked to the effector domains of Pseudomonas aeruginosa exotoxin A. Purified YC15-PE38 displayed highly selective and potent killing of a gH-expressing transfectant cell line (subnanomolar IC(50)). The immunotoxin also strongly inhibited production of infectious KSHV virions from an induced chronically infected cell line, by virtue of selective killing of the virus-producing cells. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir. These results provide support for the development of anti-KSHV strategies based on targeted killing of infected cells expressing lytic phase genes.

Previously, we identified that both human herpesvirus 6 and human immunodeficiency virus type 1 Tat were important cofactors that activated lytic cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we further investigated the potential of herpes simplex virus type 1 (HSV-1) to influence KSHV replication. We demonstrated that HSV-1 was a potentially important factor in the pathogenesis of Kaposi's sarcoma, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV ORF50 and a luciferase reporter assay testing ORF50 promoter-driven luciferase activity. Finally, we discovered that production of human interleukin-10 (IL-10) and IL-4 partially contributed to HSV-1-induced KSHV replication. Our data present the first direct evidence that HSV-1 can activate KSHV lytic replication and suggest a role of HSV-1 in KSHV pathogenesis.

The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52 -null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52 -null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.

gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.

The Kaposi’s sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation., Author Summary During viral replication, the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) modulates both host and viral gene expression. KSHV ORF57 is a multifunctional posttranscriptional regulator that is essential for viral replication and stabilizes viral RNAs. Previous studies demonstrated that ORF57 RNA-binding is essential for its activity, but the full spectrum of ORF57 targets are unknown. Here we employed a high-throughput analysis to identify RNA fragments bound by ORF57 during lytic reactivation. As expected, we found targets that mapped to the viral genome, and we further uncovered novel host targets, a subset of which had ORF57 bound near their 5´ ends. Further examination of this subset demonstrated that ORF57 bound preferentially at the 5´-most exon-intron boundary. ORF57 affected the pre-mRNA abundance from these genes, most likely by stabilizing otherwise unstable inefficiently spliced pre-mRNAs. In at least one case, this stabilization led to increases in mRNA expression of the host gene. We suggest that KSHV employs the same mechanism to stabilize intronless viral RNAs and cellular unspliced pre-mRNAs to modulate viral and host gene expression during lytic reactivation.

The ORF57 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a nuclear protein expressed during the lytic phase of KSHV replication. An ORF57 homolog is present in all known human herpesviruses and many animal herpesviruses. Many of these proteins have been demonstrated to have essential transcriptional and posttranscriptional regulatory functions. ORF57 enhances expression of reporter genes posttranscriptionally in vitro and may synergize with transcription factors to enhance gene transcription. However, the biologic role of ORF57 in KSHV replication has not been established. In this study, we demonstrate that ORF57 is essential for productive KSHV lytic replication by constructing a recombinant KSHV in which ORF57 expression has been specifically inactivated. The ORF57-null KSHV recombinant was unable to produce virion progeny or fully express several other lytic KSHV genes except when ORF57 was provided in trans . The Epstein-Barr virus (EBV) homolog of ORF57, SM, was unable to rescue lytic KSHV virion production, although EBV SM does enhance KSHV lytic gene expression from the ORF57-null mutant. Conversely, ORF57 did not rescue an SM-null recombinant EBV, indicating the existence of virus-specific functions for the ORF57 family of genes.

Human herpesvirus-8 (HHV-8) is aetiologically linked to Kaposi's sarcoma and primary effusion lymphoma. Although interferon-α(IFN-α) and interferon-γ(IFN-γ) are both antiviral cytokines, IFN-αblocks entry of HHV-8 into the lytic phase, whereas IFN-γinduces an increase in the percentage of cells undergoing lytic replication. Multiple events in the lytic cascade must be completed to produce infectious virus. The ability of both types of IFN to affect the production of infectious virus was explored. Both IFN-αand IFN-γinduced expression of the antiviral proteins double-stranded RNA-activated protein kinase (PKR) and 2′5′-oligoadenylate synthetase (2′5′-OAS) in HHV-8-infected BCBL-1 cells. Higher levels resulted from incubation with IFN-αthan with IFN-γ, whereas IFN-γinduced higher levels of IRF-1 than did IFN-α. IFN-γinduced a minor increase in lytic viral gene expression, which was not accompanied by a detectible increase in infectious virus. When lytic replication of HHV-8 was induced using TPA, high levels of infectious virus appeared in the conditioned medium. When IFN-γwas present during TPA stimulation, the production of infectious virus was reduced by at least a 60 %, and IFN-αfully blocked TPA-induced production of infectious virus. The greater reduction of viral production that occurred with IFN-αis consistent with the higher levels of the antiviral proteins PKR and 2′5′-OAS induced by IFN-αthan by IFN-γ. These studies indicate that the augmentation of cellular antiviral defences by IFN-γwas sufficient to prevent production of infectious virus despite IFN-γ-induced entry of some cells into the lytic phase of HHV-8 replication.

Human herpesvirus 8 (HHV-8) encodes multiple proteins that disrupt the host antiviral response, including viral interferon (IFN) regulatory factor 1 (vIRF-1). The product of the vIRF-1 gene blocks responses to IFN when overexpressed by transfection, but the functional consequence of vIRF-1 that is expressed during infection with HHV-8 is not known. These studies demonstrate that BCBL-1 cells that were latently infected with HHV-8 expressed low levels of vIRF-1 that were associated with PML bodies, whereas much higher levels of vIRF-1 were transiently expressed during the lytic phase of HHV-8 replication. The low levels of vIRF-1 that were associated with PML bodies were insufficient to block alpha IFN (IFN-α)-induced alterations in gene expression, whereas cells that expressed high levels of vIRF-1 were resistant to some changes induced by IFN-α, including the expression of the double-stranded-RNA-activated protein kinase. High levels of vIRF-1 were expressed for only a short period during the lytic cascade, so many cells with HHV-8 in the lytic phase responded to IFN-α with increased expression of antiviral genes and enhanced apoptosis. Furthermore, the production of infectious virus was severely compromised when IFN-α was present early during the lytic cascade. These studies indicate that the transient expression of high levels of vIRF-1 is inadequate to subvert many of the antiviral effects of IFN-α so that IFN-α can effectively induce apoptosis and block production of infectious virus when present early in the lytic cascade of HHV-8.