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Antibodies are powerful tools to detect expressed proteins. However off-target recognition can confound their use. Therefore, careful characterization is needed to validate specificity in distinct applications. Here we report the sequence and characterization of a mouse recombinant antibody that specifically detects ORF46 of murine gammaherpesvirus 68 (MHV68). This ORF encodes the viral uracil DNA glycosylase (vUNG). The antibody does not recognize murine uracil DNA glycosylase and is useful in detecting vUNG expressed in virally infected cells. It can detect expressed vUNG in cells via immunostaining and microscopy or flow cytometry analysis. The antibody can detect vUNG from lysates of expressing cells via immunoblot under native conditions but not denaturing conditions. This suggests it recognizes a confirmational based epitope. Altogether this manuscript describes the utility of the anti-vUNG antibody and suitability for use in studies of MHV68 infected cells.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells ofCD19cre/+Stat3f/fmice reduced peak latency approximately 7-fold. However, infectedCD19cre/+Stat3f/fmice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to 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 chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, 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 STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight 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 gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor 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 key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Gammaherpesviruses are an important class of oncogenic pathogens that are exquisitely evolved to their respective hosts. As such, the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) do not naturally infect nonhuman primates or rodents. There is a clear need to fully explore mechanisms of gammaherpesvirus pathogenesis, host control, and immune evasion in the host. A gammaherpesvirus pathogen isolated from murid rodents was first reported in 1980; 40 years later, murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68) infection of laboratory mice is a well-established pathogenesis system recognized for its utility in applying state-of-the-art approaches to investigate virus-host interactions ranging from the whole host to the individual cell. Here, we highlight recent advancements in our understanding of the processes by which MHV68 colonizes the host and drives disease. Lessons that inform KSHV and EBV pathogenesis and provide future avenues for novel interventions against infection and virus-associated cancers are emphasized.

SUMMARYHerpesviruses express over 100 transcripts, including messenger RNAs, long noncoding RNAs, and microRNAs. Herein, we add to this list by performing circular RNA (circRNA) profiling for Herpes Simplex Virus-1, Kaposi sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68. Using cell culture and animal models of lytic and latent infection, we identified thousands of back-splicing variants. This included circRNAs derived from viral latency genes (circK12, circLAT). During lytic infection circRNAs tiled the viral genome, with a transcriptional density 4,500-fold greater than the host. We characterized cis- and trans-elements controlling back-splicing and found that lytic infection promoted rampant, sequence-independent back-splicing. Using eCLIP and 4SU-Sequencing, we determined that the KSHV RNA binding protein (ORF57) preferentially bound viral circRNAs and globally promoted accumulation post-transcriptionally. Our work elucidates a unique splicing mechanism driven by late lytic replication and identifies a class of transcripts with potential to function in replication, persistence, or tumorigenesis.

Noncanonical NF-κB signaling is activated in B cells via the tumor necrosis factor (TNF) receptor superfamily members CD40, lymphotoxin β receptor (LTβR), and B-cell-activating factor receptor (BAFF-R). The noncanonical pathway is required at multiple stages of B cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt noncanonical NF-κB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IκB kinase α (IKKα), named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NF-κB-inducing kinase (NIK). We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTβR-mediated activation of NF-κB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in the colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and wild-type (WT) MHV68 at 16 days postinfection (dpi). Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the noncanonical NF-κB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68. IMPORTANCE The latency programs of the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block noncanonical NF-κB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKα that is nonresponsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the noncanonical NF-κB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.

Kaposi sarcoma (KS), caused by Kaposi sarcoma herpesvirus (KSHV), is a multicentric tumor characterized by abnormal vasculature and proliferation of KSHV-infected spindle cells. KS commonly involves the skin but in severe cases KS can also involve the gastrointestinal tract (GI). Here, we sought to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions. Skin and GI KS were compared to normal matched samples using bulk RNA sequencing.Twenty-two paired samples of KS and normal tissue were obtained (skin (10 pairs) and GI (12 pairs)) from 19 patients with KS of whom 17 had concurrent HIV infection. Seven paired samples were from patients who had received prior KS therapy. Three patients provided both skin and GI samples at the same timepoint. These analyses identified 370 differentially expressed genes unique to cutaneous KS and 58 DEGs unique to GI KS compared to normal skin or GI tissues. Twenty-six differentially expressed genes overlapped between skin and GI KS, which included FLT4, which encodes for a VEGF-C and VEGF-D receptor, and STC1. KSHV infection of primary lymphatic endothelial cells (LECs) resulted in increased angiogenesis, and repression of STC1 or FLT4 inhibited angiogenesis. The analyses of KSHV expression from KS lesions identified certain lytic genes, specifically ORF75, that were consistently expressed, and these expression patterns differed from laboratory infection of LECs with KSHV and KSHV gene expression in PEL cell lines. This study demonstrates that complex patterns of gene expression are found in KS tissue that differ from the canonical latent/lytic programs seen in KSHV cell lines and also demonstrates differences in viral gene and clinically relevant host gene expression in skin and GI KS that may offer insights into the pathogenesis of these forms of KS.One sentence summaryKaposi sarcoma that manifests in the skin and gastrointestinal tracts differ by human and viral gene expression.

Non-canonical NF-kappaB signaling is activated in B cells via TNF receptor superfamily members CD40, Lymphotoxin beta-R, and BAFF-R. The non-canonical pathway is required at multiple stages of B-cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt non-canonical NF-kappaB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IKKalpha, named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NIK. We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTbetaR-mediated activation of NF-kappaB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and WT MHV68 at 16 dpi. Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the non-canonical NF-kappaB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68.IMPORTANCEThe latency programs of the human gammaherpesviruses EBV and KSHV are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block non-canonical NF-kappaB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKalpha that is non-responsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the non-canonical NF-kappaB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.

We have developed a novel oxidized graphene nanoribbon-based platform (O-GNR) for gene delivery of double-stranded DNA into mammalian cells. O-GNRs, synthesized via longitudinal unzipping of multi-walled carbon nanotubes (MWCNTs), exhibited efficient DNA loading of small dsDNA fragments. Fourier Transform Infrared Spectroscopy identified stretching peaks in the O-P-O and DNA sugar phosphate backbone that were consistent with DNA loading onto O-GNRs. The presence of salts in the loading buffer promoted DNA loading and effective dispersion of O-GNRs. DNA:O-GNR complexes were stable upon treatment with surfactants Tween 20 and Triton-X100. O-GNRs did not impact the viability of mammalian cells. Last, the detection of GFP expression upon transfection of the DNA:O-GNR complex indicated that the cargo DNA is expressed in the nucleus. Taken together, O-GNRs function as a platform for gene delivery to mammalian cells.

Germinal center B cells infected with gammaherpesvirus display altered repertoire with biased usage of lambda light chain and skewed utilization of IGHV genes., The gammaherpesviruses (γHVs), human Kaposi sarcoma-associated herpesvirus (KSHV), EBV, and murine γHV68 are prevalent infections associated with lymphocyte pathologies. After primary infection, EBV and γHV68 undergo latent expansion in germinal center (GC) B cells and persists in memory cells. The GC reaction evolves and selects antigen-specific B cells for memory development but whether γHV passively transients or manipulates this process in vivo is unknown. Using the γHV68 infection model, we analyzed the Ig repertoire of infected and uninfected GC cells from individual mice. We found that infected cells displayed the hallmarks of affinity maturation, hypermutation, and isotype switching but underwent clonal expansion. Strikingly, infected cells displayed distinct repertoire, not found in uninfected cells, with recurrent utilization of certain Ig heavy V segments including Ighv10-1. In a manner observed with KSHV, γHV68 infected cells also displayed lambda light chain bias. Thus, γHV68 subverts GC selection to expand in a specific B cell subset during the process that develops long-lived immunologic memory.

The hypoxia-inducible factor 1 alpha (HIF1α) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses (γHV), Kaposi sarcoma herpes virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIF1α during the virus life cycle and its biological relevance in the context of host has been challenging due to the lack of animal models for human γHV. To study the role of HIF1α, we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIF1α protein and HIF1α-responsive gene expression in permissive cells. siRNA silencing or drug-inhibition of HIF1α reduce virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia-responsive elements (HREs) such as the viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1α transcriptional activity during KSHV infection. We found that the promoter of MHV68 ORF74 is responsive to HIF1α and MHV-68 RTA. Moreover, Intranasal infection of HIF1αLoxP/LoxP mice with MHV68 expressing Cre- recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Low oxygen concentrations accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIF1α plays a critical role in promoting virus replication and reactivation from latency by impacting viral gene expression. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis., Author summary The host oxygen-sensing machinery, including the HIF1α pathway, is important during the viral life cycle of oncogenic gammaherpesviruses such as KSHV and EBV. However, due to the host specificity, the effects of HIF1α in herpes biology is limited to studies with in vitro systems. Here, we study the role of HIF1α using the mouse gammaherpesvirus 68 (MHV68) that readily infects laboratory mice. We demonstrate that MHV68 infection upregulates HIF1α during replication and inactivation of HIF1α transcriptional activity significantly decreased viral gene expression, which results in impaired virus production in vitro. In vivo deletion of HIF1α impaired viral expansion during acute infection and affected reactivation from latency. These results show the importance of the interplay with the oxygen-sensing machinery in gammaherpesvirus infection and pathogenesis, placing the MHV68 mouse model as a unique platform to gain insight into this important aspect of oncogenic gammaherpesviruses biology and to test HIF1α targeted therapeutics.

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

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.

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Gammaherpesviruses establish lifelong infections that are associated with the development of cancer. These viruses subvert many aspects of the innate and adaptive immune response of the host. The inflammasome, a macromolecular protein complex that controls inflammatory responses to intracellular danger signals generated by pathogens, is both activated and subverted during human gammaherpesvirus infection in culture. The impact of the inflammasome response on gammaherpesvirus replication and latency in vivo is not known. Caspase-1 is the inflammasome effector protease that cleaves the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. We infected caspase-1 -deficient mice with murine gammaherpesvirus 68 (MHV68) and observed no impact on acute replication in the lung or latency and reactivation from latency in the spleen. This led us to examine the effect of viral infection on inflammasome responses in bone marrow-derived macrophages. We determined that infection of macrophages with MHV68 led to a robust interferon response but failed to activate caspase-1 or induce the secretion of IL-1β. In addition, MHV68 infection led to a reduction in IL-1β production after extrinsic lipopolysaccharide stimulation or upon coinfection with Salmonella enterica serovar Typhimurium. Interestingly, this impairment occurred at the proIL-1β transcript level and was independent of the RTA, the viral lytic r eplication and t ranscription a ctivator. Taken together, MHV68 impairs the inflammasome response by inhibiting IL-1β production during the initial stages of infection. IMPORTANCE Gammaherpesviruses persist for the lifetime of the host. To accomplish this, they must evade recognition and clearance by the immune system. The inflammasome consists of proteins that detect foreign molecules in the cell and respond by secreting proinflammatory signaling proteins that recruit immune cells to clear the infection. Unexpectedly, we found that murine gammaherpesvirus pathogenesis was not enhanced in mice lacking caspase-1 , a critical inflammasome component. This led us to investigate whether the virus actively impairs the inflammasome response. We found that the inflammasome was not activated upon macrophage cell infection with murine gammaherpesvirus 68. Infection also prevented the host cell inflammasome response to other pathogen-associated molecular patterns, indicated by reduced production of the proinflammatory cytokine IL-1β upon bacterial coinfection. Taken together, murine gammaherpesvirus impairment of the inflammatory cytokine IL-1β in macrophages identifies one mechanism by which the virus may inhibit caspase-1-dependent immune responses in the infected animal.

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.

Herpesviruses establish life-long chronic infections that place infected hosts at risk for severe disease. Herpesvirus genomes readily undergo homologous recombination (HR) during productive replication, often leading to wild-type (WT) reversion during complementation of replication-defective and attenuated viruses via HR with the helper gene provided in trans. To overcome this barrier, we developed a synthetic-biology approach based on a technique known as codon shuffling. Computer-assisted algorithms redistribute codons in a helper gene, thereby eliminating regions of homology, while enabling manipulation of factors such as codon-pair bias and CpG content to effectively titrate helper-gene protein levels. We apply this technique to rescue the replication of a murine gammaherpesvirus engineered with a mutation in the major immediate-early transactivator protein RTA. Complementation with codon-shuffled RTA constructs did not yield any WT revertant virus, a sharp contrast to WT virus contamination frequently observed during complementation with an unmodified helper gene. We further demonstrate the importance of eliminating WT virus contamination in an animal model of gammaherpesvirus lethality. We propose complementation by codon shuffling as a means to produce replication-defective or attenuated viruses. This method has immediate utility for investigating roles of essential genes in viral replication and will better enable future development of herpesvirus vaccines.

Technological advances in genome-wide transcript analysis, referred to as the transcriptome, using microarrays and deep RNA sequencing methodologies are rapidly extending our understanding of the genetic content of the gammaherpesviruses. These vast transcript analyses continue to uncover the complexity of coding transcripts due to alternative splicing, translation initiation and termination, as well as regulatory RNAs of the gammaherpesviruses. A full assessment of the transcriptome requires that our analysis be extended to the virion and exosomes of infected cells since viral and host mRNAs, miRNAs, and other non-coding RNAs seem purposefully incorporated to exert function upon delivery to naïve cells. Understanding the regulation, biogenesis and function of the recently discovered transcripts will extend beyond pathogenesis and oncogenic events to offer key insights for basic RNA processes of the cell.

The secreted M1 protein of murine gammaherpesvirus 68 (MHV68) promotes effector Vβ4 + CD8 + T cell expansion to impact virus control and immune-mediated pathologies in C57BL/6 mice, but not BALB/c mice. We report a striking increase in the number of genome-positive, IgD − B cells during chronic infection of both mouse strains. This suggests a novel role for M1 in influencing long-term maintenance in a major latency reservoir irrespective of the degree of Vβ4 + CD8 + T cell expansion.

A challenging property of gammaherpesviruses is their ability to establish lifelong persistence. The establishment of latency in B cells is thought to involve active virus engagement of host signaling pathways. Pathogenic effects of these viruses during latency or following reactivation can be devastating to the host. Many cancers, including those associated with members of the gammaherpesvirus family, Kaposi’s sarcoma-associated herpesvirus and Epstein-Barr virus, express elevated levels of active host signal transducer and activator of transcription-3 (STAT3). STAT3 is activated by tyrosine phosphorylation in response to many cytokines and can orchestrate effector responses that include proliferation, inflammation, metastasis, and developmental programming. However, the contribution of STAT3 to gammaherpesvirus pathogenesis remains to be completely understood. This is the first study to have identified STAT3 as a critical host determinant of the ability of gammaherpesvirus to establish long-term latency in an animal model of disease. Following an acute infection, murine gammaherpesvirus 68 (MHV68) established latency in resident B cells, but establishment of latency was dramatically reduced in animals with a B cell-specific STAT3 deletion. The lack of STAT3 in B cells did not impair germinal center responses for immunoglobulin (Ig) class switching in the spleen and did not reduce either total or virus-specific IgG titers. Although ablation of STAT3 in B cells did not have a global effect on these assays of B cell function, it had long-term consequences for the viral load of the host, since virus latency was reduced at 6 to 8 weeks postinfection. Our findings establish host STAT3 as a mediator of gammaherpesvirus persistence., IMPORTANCE The insidious ability of gammaherpesviruses to establish latent infections can have detrimental consequences for the host. Identification of host factors that promote viral latency is essential for understanding latency mechanisms and for therapeutic interventions. We provide the first evidence that STAT3 expression is needed for murine gammaherpesvirus 68 to establish latency in primary B cells during an active immune response to infection. STAT3 deletion in B cells does not impair adaptive immune control of the virus, but loss of STAT3 in B cells has a long-lasting impact on viral persistence. These results indicate a potential therapeutic benefit of STAT3 inhibitors for combating gammaherpesvirus latency and, thereby, associated pathologies.

Herpesviruses establish a chronic infection in the host characterized by intervals of lytic replication, quiescent latency, and reactivation from latency. Murine gammaherpesvirus 68 (MHV68) naturally infects small rodents and has genetic and biologic parallels with the human gammaherpesviruses (gHVs), Kaposi’s sarcoma-associated herpesvirus and Epstein–Barr virus. The murine gammaherpesvirus model pathogen system provides a platform to apply cutting-edge approaches to dissect the interplay of gammaherpesvirus and host determinants that enable colonization of the host, and that shape the latent or lytic fate of an infected cell. This knowledge is critical for the development of novel therapeutic interventions against the oncogenic gHVs. The nuclear factor kappa B (NF-κB) signaling pathway is well-known for its role in the promotion of inflammation and many aspects of B cell biology. Here, we review key aspects of the virus lifecycle in the host, with an emphasis on the route that the virus takes to gain access to the B cell latency reservoir. We highlight how the murine gammaherpesvirus requires components of the NF-κB signaling pathway to promote replication, latency establishment, and maintenance of latency. These studies emphasize the complexity of gammaherpesvirus interactions with NF-κB signaling components that direct innate and adaptive immune responses of the host. Importantly, multiple facets of NF-κB signaling have been identified that might be targeted to reduce the burden of gammaherpesvirus-associated diseases.

The human roseoloviruses, human herpesviruses 6A (HHV-6A), HHV-6B, and HHV-7, are highly prevalent viruses that typically cause fever/rash illnesses such as roseola during early life primary infections. They also cause significant neurologic disease and complications following stem cell and solid organ transplantation, and have suggestive but less certain etiologic associations with other neurologic diseases and immunologic disorders. The US National Institute of Allergy and Infectious Diseases recently sponsored a workshop (Roseoloviruses: Clinical Impact, Interventions, and Research Needs) to discuss disease associations, novel biology, and the many unmet research needs related to Roseoloviruses. This perspective is a distillation of the workshop's presentations and discussions, with a focus on the more general research priorities that emerged.

Gammaherpesviruses (γHV) are oncogenic viruses that establish lifelong latency in B cells. Chronic infection with human γHV, Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) is associated with the development of lymphoproliferative diseases, lymphomas and other types of cancer. The murine equivalent of human γHV, MHV68, is widely used as an animal model for γHV infection. Primary infection is associated with mucosal tissue replication, expansion in germinal center (GC) B cells and chronic persistence in host’s memory B cells. The GC compartment undergoes somatic hypermutation (SHM), class switch recombination (CSR) and repertoire selection which generate long lived antibody-secreting plasma cells and memory B cells. It is not clear what impact γHV infection has on B cell differentiation, affinity maturation and clonal proliferation. We isolated and comparatively analyzed the B cell repertoire of infected and non-infected GC cells from individual mice. This was done by sequencing repertoire from both the single cells and GC populations using NGS high-throughput sequencing. We determined the effect of infection on diversity, selection, mutagenesis and expansion of clones within the GC population of a mouse. Our data indicate that repertoire from MHV68 infected B cell are distinct from non-infected GC cells and is characterized by unique clones, repertoire bias and inappropriate expansion of certain clones. Moreover, both B cell populations share CDR3 variants only on minimum level. Thus, our findings demonstrate that viral infection cause a subversion of GC mutagenic and selective processes which result in a skewed repertoire.

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host., Author summary Gammaherpesviruses are infectious agents that cause cancer. The study of viral genes unique to this subfamily may offer insight into the strategies that these viruses use to persist in the host and drive disease. The vFGARATs are a family of viral proteins found only in gammaherpesviruses, and are critical for replication in cell culture. Here we report that a rhadinovirus of rodents requires a previously uncharacterized vFGARAT family member, ORF75A, to support viral growth and persistence in mice. In addition, viruses lacking ORF75A are defective in the production of infectious viral particles. Thus, duplications and functional divergence of the various vFGARATs in the rhadinovirus lineage have likely been driven by selective pressures to disseminate within and colonize the host. Identification of the shared host processes that are targeted by the diverse family of vFGARATs may reveal novel targets for therapeutic agents to prevent life-long infections by these oncogenic viruses.

Little is known about herpesvirus modulation of T cell activation in latently infected individuals or the implications of such for chronic immune disorders. Murine gammaherpesvirus 68 (MHV68) elicits persistent activation of CD8(+) T cells bearing a Vbeta4(+) T cell receptor (TCR) by a completely unknown mechanism. We show that a novel MHV68 protein encoded by the M1 gene is responsible for Vbeta4(+) CD8(+) T cell stimulation in a manner reminiscent of a viral superantigen. During infection, M1 expression induces a Vbeta4(+) effector T cell response that resists functional exhaustion and appears to suppress virus reactivation from peritoneal cells by means of long-term interferon-gamma (IFNgamma) production. Mice lacking an IFNgamma receptor (IFNgammaR(-/-)) fail to control MHV68 replication, and Vbeta4(+) and CD8(+) T cell activation by M1 instead contributes to severe inflammation and multiorgan fibrotic disease. Thus, M1 manipulates the host CD8(+) T cell response in a manner that facilitates latent infection in an immunocompetent setting, but promotes disease during a dysregulated immune response. Identification of a viral pathogenecity determinant with superantigen-like activity for CD8(+) T cells broadens the known repertoire of viral immunomodulatory molecules, and its function illustrates the delicate balance achieved between persistent viruses and the host immune response.

The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation.

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.

Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (ΔUNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68ΔUNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCE Herpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the initial site of acute replication was associated with a substantial delay of latency establishment in the spleen. The levels of host UNG were substantially lower in the lung compared to the spleen, suggesting that herpesviruses encode a viral UNG to compensate for reduced host enzyme levels in some cell types and tissues. These data suggest that intervention at the site of initial replicative expansion can delay the establishment of latency, a hallmark of chronic herpesvirus infection.

Human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, and HHV-7) are classified within the roseolovirus genus of the betaherpesvirus subfamily. Most humans likely harbor at least two of these large DNA viruses, and 1% of humans harbor germline chromosomally integrated (ci) HHV-6A or HHV-6B genomes. Differences at the genetic level manifest as distinct biologic properties during infection and disease. We provide a brief synopsis of roseolovirus replication and highlight the unique properties of their lifecycle and what is known about the viral gene products that mediate these functions. In the nearly 30 years since their discovery, we have only begun to unlock the molecular strategies these highly evolved pathogens employ to establish and maintain chronic infections in humans.

The Roseolovirus genus of the Betaherpesvirinae consists of the very closely related viruses, human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) plus the somewhat more distantly related human herpesvirus 7 (HHV-7). The roseoloviruses each encode a homolog of the alphaherpesvirus origin binding protein (OBP) which is required for lytic DNA replication. In contrast, members of the other betaherpesvirus genera, the cytomegaloviruses, initiate DNA replication by a different mechanism. To better understand the basis of roseolovirus OBP sequence specificity, we investigated their ability to recognize each other's binding sites. HHV-6A OBP (OBP H6A ) and HHV-6B OBP (OBP H6B ) each bind to both of the HHV-7 OBP sites (OBP-1 and OBP-2) with similar strengths, which are also similar to their nearly equivalent interactions with their own sites. In contrast, HHV-7 OBP (OBP H7 ) had a gradient of binding preferences: HHV-7 OBP-2 > HHV-6 OBP-2 > HHV-7 OBP-1 > HHV-6 OBP-1. Thus, the roseolovirus OBPs are not equally reciprocal in their recognition of each other's OBP sites, suggesting that the sequence requirements for the interaction of OBPH7 at the OBP sites in its cognate ori Lyt differ from those of OBPH6A and OBPH6B.

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication ( ori Lyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP H7 ) with its cognate sites in the 600-bp HHV-7 ori Lyt. We expressed the carboxyl-terminal domain of OBP H7 and found that amino acids 484 to 787 of OBP H7 were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP H7 has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP H6B ), which binds with similar affinity to its two cognate OBP sites in the HHV-6B ori Lyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP H7 consensus recognition sequence of the 9-bp core, BRT YCWCC T (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP H6B consensus YGW YCWCC Y and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP H7 interacts along one face of the DNA helix, with the major groove, as do OBP H6B and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP H7 and OBP H6B .

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B, respectively) encode homologs (U94) of the parvovirus nonstructural gene, ns1 or rep. Here we describe the HHV-6B homolog and analyze its genetic heterogeneity and transcription. U94 nucleotide and amino acid sequences differ by approximately 3.5% and 2.5%, respectively, between HHV-6A and HHV-6B. Among a collection of 17 clinically and geographically disparate HHV-6 isolates, intravariant nucleotide and amino acid sequence divergence was less than 0.6% and 0.2%, respectively; all 13 HHV-6B isolates had identical amino acid sequences. The U94 transcript is spliced to remove a 2.6-kb intron and is expressed at very low levels relative to other HHV-6B genes, reaching approximately 10 copies per cell 3 days after infection. The mRNA has several small AUG-initiated open reading frames upstream of the U94 open reading frame, a hallmark of proteins expressed at low levels. Consistent with this, the U94-encoded protein was immunologically undetectable in HHV-6B-infected cells. The high degree of sequence conservation suggests that the gene function is nearly intolerant of sequence variation. The low abundance of U94 transcripts and the presence of encoded inefficient translation initiation suggest that the U94 protein may be required only in small amounts during infection.

Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX) activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression., Author Summary Many viruses restrict host gene expression during infection, presumably to provide a competitive expression advantage to viral transcripts. Not surprisingly, viruses that induce this ‘host shutoff’ phenotype therefore generally possess mechanisms to selectively spare viral genes. Gammaherpesviruses promote host shutoff by inducing widespread mRNA degradation, a process initiated by the viral SOX nuclease. However, the effect of SOX on viral mRNA during infection was unknown. Here, we reveal that during infection with the murine gammaherpesvirus MHV68, the majority of viral transcripts of all kinetic classes are broadly down regulated through the activity of the MHV68 SOX protein (muSOX). We further demonstrate that in the absence of muSOX-induced control of viral mRNA abundance, viral protein levels increase, thereby affecting the composition of progeny viral particles. Altered virion composition directly impacts early events such as entry and induction of lytic gene expression in subsequent rounds of replication. Furthermore, decreasing both virus and host gene expression via global mRNA degradation is critical for viral replication in a cell type specific manner both in vitro and in vivo. This is the first example of a eukaryotic virus whose host shutoff mechanism similarly tempers viral gene expression, and highlights the degree to which gammaherpesviral gene expression must be fine tuned to ensure replicative success.

The human gammaherpesviruses establish life-long infections that are associated with the development of lymphomas and neoplasms, especially in immunocompromised individuals. T cells play a crucial role in the control of gammaherpesvirus infection through multiple functions, including the direct killing of infected cells, production of cytokines such as interferon-γ (IFN-γ), and costimulation of B cells. Impaired T cell function in mice infected with murine gammaherpesvirus 68 (MHV68) leads to increased reactivation and pathologies, including a higher incidence of lymphoid hyperplasia. Here we report that the absence of Suppressor of TCR signaling -1 and -2 (Sts-1(-/-)/2(-/-)) during MHV68 infection leads to the generation of T cells with significantly heightened responses. Transient differences in the T and B cell response of infected Sts-1(-/-)/2(-/-) (Sts dKO) mice were also observed when compared to WT mice. However, these alterations in the immune response and the overall absence of Sts-1 and Sts-2 did not impact viral pathogenesis or lead to pathology. Acute lytic replication in the lungs, establishment of latency in the spleen and reactivation from latency in the spleen in the Sts dKO mice were comparable to WT mice. Our studies indicate that Sts-1 and Sts-2 are not required for the immune control of MHV68 in a normal course of gammaherpesvirus infection, but suggest that interference with negative regulators of T cell responses might be further explored as a safe and efficacious strategy to improve adoptive T cell therapy.

Humans encode seven APOBEC3 (A3A-A3H) cytidine deaminase proteins that differ in their expression profiles, preferred nucleotide recognition sequence and capacity for restriction of RNA and DNA viruses. We identified APOBEC3 hotspots in numerous herpesvirus genomes. To determine the impact of host APOBEC3 on herpesvirus biology in vivo, we examined whether murine APOBEC3 (mA3) restricts murine gammaherpesvirus 68 (MHV68). Viral replication was impaired by several human APOBEC3 proteins, but not mA3, upon transfection of the viral genome. The restriction was abrogated upon mutation of the A3A and A3B active sites. Interestingly, virus restriction by A3A, A3B, A3C, and A3DE was lost if the infectious DNA was delivered by the virion. MHV68 pathogenesis, including lung replication and splenic latency, was not altered in mice lacking mA3. We infer that mA3 does not restrict wild type MHV68 and restriction by human A3s may be limited in the herpesvirus replication process.

The incidence of idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPF and γherpesvirus infection, we infected young (2-3 mo) and old (≥18 mo) C57BL/6 mice with the murine γherpesvirus 68. Acute murine γherpesvirus 68 infection in aging mice resulted in severe pneumonitis and fibrosis compared with young animals. Progressive clinical deterioration and lung fibrosis in the late chronic phase of infection was observed exclusively in old mice with diminution of tidal volume. Infected aging mice showed higher expression of transforming growth factor-β during the acute phase of infection. In addition, aging, infected mice showed elevation of proinflammatory cytokines and the fibrocyte recruitment chemokine, CXCL12, in bronchoalveolar lavage. Analyses of lytic virus infection and virus reactivation indicate that old mice were able to control chronic infection and elicit antivirus immune responses. However, old, infected mice showed a significant increase in apoptotic responses determined by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay, levels of caspase-3, and expression of the proapoptotitc molecule, Bcl-2 interacting mediator. Apoptosis of type II lung epithelial cells in aging lungs was accompanied by up-regulation of endoplasmic reticulum stress marker, binding immunoglobulin protein, and splicing of X-box-binding protein 1. These results indicate that the aging lung is more susceptible to injury and fibrosis associated with endoplasmic reticulum stress, apoptosis of type II lung epithelial cells, and activation of profibrotic pathways.

We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5′ rapid amplification of cDNA ends. The ∼1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection.

Four human herpesviruses were discovered in a eight-year period between 1986 and 1994. This exciting era of virus discovery was driven in part by the search for HIV and HIV-related diseases coupled with the development of new molecular tools such as PCR, automated Sanger sequencing, and subtractive hybridization. Three of these viruses, human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B), and human herpesvirus 7 (HHV-7), were initially cultured from peripheral blood mononuclear cells. All three were found to be T lymphotropic viruses that were most closely related to human cytomegalovirus (HCMV), placing them in the betaherpesvirus family. Given their tight biologic and genetic relationships and clear etiologic link to roseola infantum, these viruses are now designated Roseoloviruses. Human herpesvirus 8 (also known as Kaposi’s sarcoma associated herpesvirus) was identified as a new member of the gammaherpesviruses, in the rhadinovirus genus. Given its clear link to HIV-related malignancies, HHV-8 research exploded.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus and IPF. Based on those observations, we have developed a mouse model in which interferon (IFN)gammaR(-/-) mice infected intranasally with murine gammaherpesvirus 68 (MHV68) develop lung fibrosis. We hypothesize that viral load was a critical factor for the development of fibrosis. Because nuclear factor (NF)-kappaB signaling is required to efficiently establish gammaherpesvirus, latency we infected IFNgammaR(-/-) mice with a MHV68 virus that expresses a mutant dominant inhibitor of the NF-kappaB signaling pathway, called IkappaBalphaM. Striking differences were observed at the onset of the chronic infection, which correlated with a decreased virus load in mice infected with MHV68-IkappaBalphaM compared with mice infected with control MHV68 (MHV68-MR). IFNgammaR(-/-) mice infected with MHV68-IkappaBalphaM lacked vasculitis and fibrosis 15 to 120 days post infection. Inhibition of NF-kappaB in MHV68-infected cells of the lungs diminished the expression of the fibrocyte recruiting chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL12, ameliorated macrophage expression of markers of alternative activation, and failed to increase expression of the integrin alphavbeta6, which is implicated in the activation of the profibrotic factor TGF-beta. Thus, the inhibition of NF-kappaB signaling in the infected lung cells of IFNgammaR(-/-) mice reduces virus persistence and ameliorates profibrotic events. Host determinants of latency might therefore represent new therapeutic targets for gammaherpesvirus-associated pulmonary fibrosis.

NF-κB signaling is critical to the survival and transformation of cells infected by the human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here we have examined how elimination of the NF-κB transcription factor p50 from mice affects the life cycle of murine gammaherpesvirus 68 (MHV68). Notably, mice lacking p50 in every cell type were unable to establish a sufficiently robust immune response to control MHV68 infection, leading to high levels of latently infected B cells detected in the spleen and persistent virus replication in the lungs. The latter correlated with very low levels of virus-specific immunoglobulin G (IgG) in the infected p50−/−mice at day 48 postinfection. Because the confounding impact of the loss of p50 on the host response to MHV68 infection prevented a direct analysis of the role of this NF-κB family member on MHV68 latency in B cells, we generated and infected mixed p50+/+/p50−/−bone marrow chimeric mice. We show that the chimeric mice were able to control acute virus replication and exhibited normal levels of virus-specific IgG at 3 months postinfection, indicating the induction of a normal host immune response to MHV68 infection. However, in p50+/+/p50−/−chimeric mice the p50−/−B cells exhibited a significant defect compared to p50+/+B cells in supporting MHV68 latency. In addition to identifying a role for p50 in the establishment of latency, we determined that the absence of p50 in a subset of the hematopoietic compartment led to persistent virus replication in the lungs of the chimeric mice, providing evidence that p50 is required for controlling virus reactivation. Taken together, these data demonstrate that p50 is required for immune control by the host and has distinct tissue-dependent roles in the regulation of murine gammaherpesvirus latency during chronic infection.