Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Open reading frames M12/M13 jointly contribute to MHV-68 latency.

Murine gammaherpesvirus 68 (MHV-68), a widely used small-animal model for the analysis of gammaherpesvirus pathogenesis, encodes the MHV-68-specific ORFs M12 and M13. The function of M12 and M13 has not been investigated so far. Therefore, we constructed and analysed recombinant MHV-68 with mutations in either M12, M13 or M12/M13. Both the M12 and M13 mutants did not display any phenotype in vitro or in vivo. However, although the M12/13 double mutant showed similar lytic growth in fibroblasts in vitro and in the lungs of infected mice as wild-type MHV-68, it was significantly attenuated in vivo during latency. This phenotype was completely restored in a revertant of the M12/13 double mutant. Thus, it appears that M12 and M13 might have redundant functions that are only revealed if both genes are lacking. The observation that M12/13 have a function during latency not only contributes to the further understanding of the pathogenesis of MHV-68 infection but might also be of interest considering that M12/13 are located at a genomic position similar to that of LMP2A and K15. The latter are important proteins of their respective human gammaherpesviruses EBV and KSHV that contribute to cellular survival, cell activation and proliferation, which was deduced from in vitro studies.

Amino acids 1811-1960 of myosin heavy chain 9 is involved in murine gammaherpesvirus 68 infection.

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 µg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811-1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.

The Interaction between Tegument Proteins ORF33 and ORF45 Plays an Essential Role in Cytoplasmic Virion Maturation of a Gammaherpesvirus.

Tegument, which occupies the space between the nucleocapsid and the envelope, is a unique structure of a herpesvirion. Tegument proteins are major components of tegument and play critical roles in virus life cycle. Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus subfamily, is closely related to two human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We have previously shown that MHV-68 ORF33, conserved among all herpesviruses, encodes a tegument protein that is associated with intranuclear capsids and is essential for virion morphogenesis and egress. Another tegument protein ORF45, which is conserved only among gammaherpesviruses, also plays an essential role in virion morphogenesis of MHV-68. In this study, we investigated the underlying mechanism and showed that these two proteins colocalize and interact with each other during virus infection. We mapped the ORF33-interacting domain to the conserved carboxyl-terminal 23 amino acids (C23) of ORF45. Deletion of the C23 coding sequence in the context of viral genome abolished the production of infectious virions. Transmission electron microscopy results demonstrated that C23 of ORF45 are essential for virion tegumentation in the cytoplasm. We further mapped the ORF45-interacting domain to the N-terminal 17 amino acids (N17) of ORF33. Deletion of the N17 coding sequence in the context of viral genome also abolished production of infectious virions, and N17 of ORF33 are also essential for virion tegumentation in the cytoplasm. Taken together, our data strongly indicate that the interaction between ORF45 and ORF33 plays an essential role in cytoplasmic maturation of MHV-68 virions. IMPORTANCE A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens. A herpesvirus particle comprises, from inside to outside, four layers: DNA core, capsid, tegument, and envelope. The tegument layer contains dozens of virally encoded tegument proteins, which play critical roles in virus assembly. Murine gammaherpesvirus 68 (MHV-68) is a tumor-associated herpesvirus and is closely related to Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. We previously found that the absence of either tegument protein ORF33 or ORF45 inhibits the translocation of nucleocapsids to the cytoplasm and blocks virion maturation, but the underlying mechanism remained unclear. Here, we showed that ORF33 interacts with ORF45. We mapped their interaction domains and constructed viral mutants with defects in ORF33-ORF45 interaction. Transmission electron microscopy data demonstrated that the assembly of these viral mutants in the cytoplasm is blocked. Our results indicate that ORF33-ORF45 interaction is essential for gammaherpesvirus replication.

A Polymorphism in the Epstein-Barr Virus EBER2 Noncoding RNA Drives In Vivo Expansion of Latently Infected B Cells.

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4), are associated with numerous malignancies, including B cell lymphomas and nasopharyngeal carcinoma. These viruses employ numerous molecular strategies to colonize the host, including the expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2, respectively) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. In work here, we used chimeric MHV68 viruses in an in vivo complementation system to test whether EBV EBER2 contributes to acute and/or chronic phases of infection. Expression of EBER2 derived from EBV strain B95-8 resulted in a significant expansion of latently infected B cells in vivo, which was accompanied by a decrease in virus-infected plasma cells. EBV strains typically carry one of two variants of EBER2, which differ primarily by a 5-nucleotide core polymorphism identified initially in the EBV strain M81. Strikingly, mutation of the 5 nucleotides that define this core polymorphism resulted in the loss of the infected B cell expansion and restored plasma cell infection. This work reveals that the B95-8 variant of EBER2 promotes the expansion of the latently infected B cell pool in vivo and may do so in part through inhibition of terminal differentiation. These findings provide new insight into mechanisms by which viral ncRNAs promote in vivo colonization and further and provide further evidence of the inherent tumorigenic risks associated with gammaherpesvirus manipulation of B cell differentiation. IMPORTANCE The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68, employ numerous strategies to colonize the host, including expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs ever identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. Work here reveals that an EBV EBER2 variant highly associated with B cell lymphoma promoted a significantly increased expansion of the infected B cell pool in vivo, which coincided with altered B cell differentiation. Mutation of the 5 nucleotides that define this EBER2 variant resulted in the loss of B cell expansion and normal B cell differentiation. These findings provide new insight into the mechanisms by which EBV manipulates B cells in vivo to retain infected cells in the high-risk B cell differentiation pathway where they are poised for tumorigenesis.

Kinetics of the Equid Herpesvirus 2 and 5 Infections among Mares and Foals from Three Polish National Studs.

Equid herpesvirus 2 (EHV-2) and 5 (EHV-5) are two γ-herpesviruses that are commonly detected from horses worldwide, based on several cross-sectional molecular surveys. Comparatively few studies examined the dynamics of γ-herpesvirus infection over time in a group of horses. The aim of the current study was to investigate the dynamics of EHV-2/5 infections among mares and their foals at three Polish national studs with different breeds of horses: Arabians, Thoroughbreds and Polish Konik horses. Nasal swabs were collected from each of 38 mare-foal pairs monthly for a period of 6 to 8 months. Virus-specific quantitative PCR assays were used to determine the viral load of EHV-2 and EHV-5 in each sample. All 76 horses sampled were positive for EHV-2 or EHV-5 on at least one sampling occasion. The majority (73/76, 96%) were infected with both EHV-2 and EHV-5. In general, the mean load of viral DNA was higher in samples from foals than from mares, but similar for EHV-2 and EHV-5 at most sampling occasions. There was, however, a considerable variability in the viral DNA load between samples collected at different times from the same foal, as well as between samples from different foals. The latter was more apparent for EHV-2 than for EHV-5. All foals became infected with both viruses early in life, before weaning, and remained positive on all, or most, subsequent samplings. The virus shedding by mares was more intermittent, indicating the existence of age-related differences. Overall, the data presented extend our knowledge of EHV-2/5 epidemiology among mares and foals.

Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposi's Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell-Specific Manner.

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma-2 herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549 cells, human foreskin fibroblasts (HFF), and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell-dependent inhibitors of KSHV and RRV infection in A549 cells and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Of note, our results indicate that recombinant IFITM overexpression may lead to results that are not representative for the situation at endogenous levels. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. Mechanistically, we found that KSHV colocalizes less with IFITM1 and IFITM2 in HUVEC than in A549 cells immediately after attachment, potentially contributing to the observed difference in restriction. IMPORTANCE IFITM proteins are the first line of defense against infection by many pathogens and may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action is well understood. We report here that in some cells but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

Olfactory Entry Promotes Herpesvirus Recombination.

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by coinfecting mice with attenuated mutants of murid herpesvirus 4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung coinfections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back toward the olfactory surface. Early infection was correspondingly focused on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. IMPORTANCE All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often coinfect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.

Birds Belonging to the Family Paridae as Another Potential Reservoir of Murine Gammaherpesvirus 68.

Ecology and epidemiology of murine gammaherpesvirus 68 (MHV-68) have been intensively studied since the isolation of the virus from murid rodents in 1976. This virus was detected in various mammalian species that share the biotope with rodent reservoirs of MHV-68. However, a survey of MHV-68 in birds has not so far been performed. Therefore, the aim of this study was to investigate the presence of MHV-68 in blood samples from two bird species captured at four localities in Slovakia. Using the nested PCR targeting ORF50 gene of MHV-68, we confirmed the presence of MHV-68 DNA in 9 out of 57 blood samples from Great tits (Parus major) (prevalence 15.8%, confidence interval [95% CI]: 8.5-27.4) and in 3 out of 43 blood samples from Eurasian blue tits (Cyanistes caeruleus) (prevalence 7.0%, 95% CI: 2.4-18.6). Our results suggest that not only mammals but also birds may serve as reservoirs for MHV-68, providing further evidence that MHV-68 is capable of frequent cross-species transmission.

Recombinant Herpesvirus Vectors: Durable Immune Responses and Durable Protection against Simian Immunodeficiency Virus SIVmac239 Acquisition.

A prophylactic vaccine that confers durable protection against human immunodeficiency virus (HIV) would provide a valuable tool to prevent new HIV/AIDS cases. As herpesviruses establish lifelong infections that remain largely subclinical, the use of persistent herpesvirus vectors to deliver HIV antigens may facilitate the induction of long-term anti-HIV immunity. We previously developed recombinant (r) forms of the gamma-herpesvirus rhesus monkey rhadinovirus (rRRV) expressing a replication-incompetent, near-full-length simian immunodeficiency virus (SIVnfl) genome. We recently showed that 8/16 rhesus macaques (RMs) vaccinated with a rDNA/rRRV-SIVnfl regimen were significantly protected against intrarectal (i.r.) challenge with SIVmac239. Here we investigated the longevity of this vaccine-mediated protection. Despite receiving no additional booster immunizations, the protected rDNA/rRRV-SIVnfl vaccinees maintained detectable cellular and humoral anti-SIV immune responses for more than 1.5 years after the rRRV boost. To assess if these responses were still protective, the rDNA/rRRV-SIVnfl vaccinees were subjected to a second round of marginal-dose i.r. SIVmac239 challenges, with eight SIV-naive RMs serving as concurrent controls. After three SIV exposures, 8/8 control animals became infected, compared to 3/8 vaccinees. This difference in SIV acquisition was statistically significant (P = 0.0035). The three vaccinated monkeys that became infected exhibited significantly lower viral loads than those in unvaccinated controls. Collectively, these data illustrate the ability of rDNA/rRRV-SIVnfl vaccination to provide long-term immunity against stringent mucosal challenges with SIVmac239. Future work is needed to identify the critical components of this vaccine-mediated protection and the extent to which it can tolerate sequence mismatches in the challenge virus. IMPORTANCE We report on the long-term follow-up of a group of rhesus macaques (RMs) that received an AIDS vaccine regimen and were subsequently protected against rectal acquisition of simian immunodeficiency virus (SIV) infection. The vaccination regimen employed included a live recombinant herpesvirus vector that establishes persistent infection in RMs. Consistent with the recurrent SIV antigen expression afforded by this herpesvirus vector, vaccinees maintained detectable SIV-specific immune responses for more than 1.5 years after the last vaccination. Importantly, these vaccinated RMs were significantly protected against a second round of rectal SIV exposures performed 1 year after the first SIV challenge phase. These results are relevant for HIV vaccine development because they show the potential of herpesvirus-based vectors to maintain functional antiretroviral immunity without the need for repeated boosting.

Myelin-specific T cells in animals with Japanese macaque encephalomyelitis.

OBJECTIVE: To determine whether animals with Japanese macaque encephalomyelitis (JME), a spontaneous demyelinating disease similar to multiple sclerosis (MS), harbor myelin-specific T cells in their central nervous system (CNS) and periphery. METHODS: Mononuclear cells (MNCs) from CNS lesions, cervical lymph nodes (LNs) and peripheral blood of Japanese macaques (JMs) with JME, and cervical LN and blood MNCs from healthy controls or animals with non-JME conditions were analyzed for the presence of myelin-specific T cells and changes in interleukin 17 (IL-17) and interferon gamma (IFNγ) expression. RESULTS: Demyelinating JME lesions contained CD4+ T cells and CD8+ T cells specific to myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and/or proteolipid protein (PLP). CD8+ T-cell responses were absent in JME peripheral blood, and in age- and sex-matched controls. However, CD4+ Th1 and Th17 responses were detected in JME peripheral blood versus controls. Cervical LN MNCs from eight of nine JME animals had CD3+ T cells specific for MOG, MBP, and PLP that were not detected in controls. Mapping myelin epitopes revealed a heterogeneity in responses among JME animals. Comparison of myelin antigen sequences with those of JM rhadinovirus (JMRV), which is found in JME lesions, identified six viral open reading frames (ORFs) with similarities to myelin antigen sequences. Overlapping peptides to these JMRV ORFs did not induce IFNγ responses. INTERPRETATIONS: JME possesses an immune-mediated component that involves both CD4+ and CD8+ T cells specific for myelin antigens. JME may shed new light on inflammatory demyelinating disease pathogenesis linked to gamma-herpesvirus infection.

Deletion of Murine Gammaherpesvirus Gene M2 in Activation-Induced Cytidine Deaminase-Expressing B Cells Impairs Host Colonization and Viral Reactivation.

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.

Viral fitness of MHV-68 viruses harboring drug resistance mutations in the protein kinase or thymidine kinase.

Murine γ-herpesvirus-68 (MHV-68), genetically and biologically related to human γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can be easily propagated in vitro allowing drug resistance studies. Previously, we described specific changes in MHV-68 protein kinase (PK) or thymidine kinase (TK) associated with resistance to various purine or pyrimidine nucleoside analogues, respectively. To investigate how specific TK and PK mutations affect viral replication capacity, we performed dual infection competition assays in which wild-type and drug-resistant virus compete in absence or presence of antivirals in Vero cells. The composition of the mixed viral population was analyzed using next-generation sequencing and relative fitness of seven MHV-68 PK or TK mutants was calculated based on the frequency of viral variants at the time of infection and after 5-days growth. A MHV-68 mutant losing the PK function due to a 2-nucleotide deletion was less fit than the wild-type virus in absence of antivirals, consistent with the essential role of viral PKs during lytic replication, but overgrew the wild-type virus under pressure of purine nucleosides. TK mutant viruses, with frameshift or missense mutations, grew equal to wild-type virus in absence of antivirals, in accordance with the viral TK function only being essential in non-replicating or in TK-deficient cells, but were more fit when treated with pyrimidine nucleosides. Moreover, TK missense mutant viruses also increased fitness under pressure of antivirals other than pyrimidine nucleosides, indicating that MHV-68 TK mutations might influence viral fitness by acting on cellular and/or viral functions that are unrelated to nucleoside activation.

Conserved Herpesvirus Kinase ORF36 Activates B2 Retrotransposons during Murine Gammaherpesvirus Infection.

Short interspersed nuclear elements (SINEs) are RNA polymerase III (RNAPIII)-transcribed, retrotransposable noncoding RNA (ncRNA) elements ubiquitously spread throughout mammalian genomes. While normally silenced in healthy somatic tissue, SINEs can be induced during infection with DNA viruses, including the model murine gammaherpesvirus 68 (MHV68). Here, we explored the mechanisms underlying MHV68 activation of SINE ncRNAs. We demonstrate that lytic MHV68 infection of B cells, macrophages, and fibroblasts leads to robust activation of the B2 family of SINEs in a cell-autonomous manner. B2 ncRNA induction requires neither host innate immune signaling factors nor involvement of the RNAPIII master regulator Maf1. However, we identified MHV68 ORF36, the conserved herpesviral kinase, as playing a key role in B2 induction during lytic infection. SINE activation is linked to ORF36 kinase activity and can also be induced by inhibition of histone deacetylases 1 and 2 (HCAC 1/2), which is one of the known ORF36 functions. Collectively, our data suggest that ORF36-mediated changes in chromatin modification contribute to B2 activation during MHV68 infection and that this activity is conserved in other herpesviral protein kinase homologs.IMPORTANCE Viral infection dramatically changes the levels of many types of RNA in a cell. In particular, certain oncogenic viruses activate expression of repetitive genes called retrotransposons, which are normally silenced due to their ability to copy and spread throughout the genome. Here, we established that infection with the gammaherpesvirus MHV68 leads to a dramatic induction of a class of noncoding retrotransposons called B2 SINEs in multiple cell types. We then explored how MHV68 activates B2 SINEs, revealing a role for the conserved herpesviral protein kinase ORF36. Both ORF36 kinase-dependent and kinase-independent functions contribute to B2 induction, perhaps through ORF36 targeting of proteins involved in controlling the accessibility of chromatin surrounding SINE loci. Understanding the features underlying induction of these elements following MHV68 infection should provide insight into core elements of SINE regulation, as well as disregulation of SINE elements associated with disease.

Membrane association of a model CD4+ T-cell vaccine antigen confers enhanced yet incomplete protection against murid herpesvirus-4 infection.

Vaccination against γ-herpesviruses has proved difficult. CD4+ T cells are essential to contain infection, but how best to prime them and whether this can reduce viral loads remain unclear. To address these questions, we used ovalbumin (OVA) as a model antigen, delivering it with murine cytomegalovirus (MCMV) to protect mice against OVA-expressing murine herpesvirus-4 (MuHV-4). Membrane-associated OVA (mOVA) was more effective than soluble OVA, both to prime CD4+ T cells and as an effector target. It was also a better target than an OVA epitope limited to infected cells, suggesting that protective CD4+ T cells recognize infected cell debris rather than infected cells themselves. While MCMV-mOVA protected acutely against MuHV-4-mOVA, long-term protection was incomplete, even when OVA-specific CD8+ T cells and B cells were also primed. Thus, even optimized single-target vaccines may poorly reduce long-term γ-herpesvirus infections.

A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses.

A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two Mamu-A*01+ RRV-naive rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real-time PCR revealed levels similar to those in monkeys infected with recombinant gL+ RRV. Comparison of RRV DNA levels in sorted CD3+ versus CD20+ versus CD14+ PBMC subpopulations indicated infection of the CD20+ subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines in vitro, in which deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks, Gag-specific CD8+ T cell responses were documented by major histocompatibility complex class I (MHC-I) tetramer staining. Vaccine-induced CD8+ T cell responses, which were predominantly directed against the Mamu-A*01-restricted Gag181-189CM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors are dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role for gL and Eph family receptors in B cell infection by RRV in vivo and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.

A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment.

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA. To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo. Using co-infection experiments and recombinant viruses, we also show that KSHV's ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.

B Cell-Intrinsic SHP1 Expression Promotes the Gammaherpesvirus-Driven Germinal Center Response and the Establishment of Chronic Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the majority of adults worldwide. Chronic gammaherpesvirus infection has been implicated in both lymphomagenesis and, somewhat controversially, autoimmune disease development. Pathogenesis is largely associated with the unique ability of gammaherpesviruses to usurp B cell differentiation, specifically, the germinal center response, to establish long-term latency in memory B cells. The host tyrosine phosphatase SHP1 is known as a brake on immune cell activation and is downregulated in several gammaherpesvirus-driven malignancies. However, here we demonstrate that B cell- but not T cell-intrinsic SHP1 expression supports the gammaherpesvirus-driven germinal center response and the establishment of viral latency. Furthermore, B cell-intrinsic SHP1 deficiency cooperated with gammaherpesvirus infection to increase the levels of double-stranded DNA-reactive antibodies at the peak of viral latency. Thus, in spite of decreased SHP1 levels in gammaherpesvirus-driven B cell lymphomas, B cell-intrinsic SHP1 expression plays a proviral role during the establishment of chronic infection, suggesting that the gammaherpesvirus-SHP1 interaction is more nuanced and is modified by the stage of infection and pathogenesis.IMPORTANCE Gammaherpesviruses establish lifelong infection in a majority of adults worldwide and are associated with a number of malignancies, including B cell lymphomas. These viruses infect naive B cells and manipulate B cell differentiation to achieve a lifelong infection of memory B cells. The germinal center stage of B cell differentiation is important as both an amplifier of the viral latent reservoir and the target of malignant transformation. In this study, we demonstrate that expression of tyrosine phosphatase SHP1, a negative regulator that normally limits the activation and proliferation of hematopoietic cells, enhances the gammaherpesvirus-driven germinal center response and the establishment of chronic infection. The results of this study uncover an intriguing beneficial interaction between gammaherpesviruses that are presumed to profit from B cell activation and a cellular phosphatase that is traditionally perceived to be a negative regulator of the same processes.