Tiled Microarray Identification of Novel Viral Transcript Structures and Distinct Transcriptional Profiles during Two Modes of Productive Murine Gammaherpesvirus 68 Infection

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Tumor suppressor p53 is activated in response to numerous cellular stresses, including viral infection. However, whether murine gammaherpesvirus 68 (MHV68) provokes p53 during the lytic replication cycle has not been extensively evaluated. Here, we demonstrate that MHV68 lytic infection induces p53 phosphorylation and stabilization in a manner that is dependent on the DNA damage response (DDR) kinase ataxia telangiectasia mutated (ATM). The induction of p53 during MHV68 infection occurred in multiple cell types, including splenocytes of infected mice. ATM and p53 activation required early viral gene expression but occurred independently of viral DNA replication. At early time points during infection, p53-responsive cellular genes were induced, coinciding with p53 stabilization and phosphorylation. However, p53-related gene expression subsided as infection progressed, even though p53 remained stable and phosphorylated. Infected cells also failed to initiate p53-dependent gene expression and undergo apoptosis in response to treatment with exogenous p53 agonists. The inhibition of p53 responses during infection required the expression of the MHV68 homologs of the shutoff and exonuclease protein (muSOX) and latency-associated nuclear antigen (mLANA). Interestingly, mLANA, but not muSOX, was necessary to prevent p53-mediated death in MHV68-infected cells under the conditions tested. This suggests that muSOX and mLANA are differentially required for inhibiting p53 in specific settings. These data reveal that DDR responses triggered by MHV68 infection promote p53 activation. However, MHV68 encodes at least two proteins capable of limiting the potential consequences of p53 function. IMPORTANCEGammaherpesviruses are oncogenic herpesviruses that establish lifelong chronic infections. Defining how gammaherpesviruses overcome host responses to infection is important for understanding how these viruses infect and cause disease. Here, we establish that murine gammaherpesvirus 68 induces the activation of tumor suppressor p53. p53 activation was dependent on the DNA damage response kinase ataxia telangiectasia mutated. Although active early after infection, p53 became dominantly inhibited as the infection cycle progressed. Viral inhibition of p53 was mediated by the murine gammaherpesvirus 68 homologs of muSOX and mLANA. The inhibition of the p53 pathway enabled infected cells to evade p53-mediated cell death responses. These data demonstrate that a gammaherpesvirus encodes multiple proteins to limit p53-mediated responses to productive viral infection, which likely benefits acute viral replication and the establishment of chronic infection. G ammaherpesviruses (GHVs) are DNA tumor viruses that include Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68), among others (1). Like all herpesviruses, GHVs exhibit two distinct infectious cycle phases, lytic replication and latency (2). The lytic cycle is the productive phase of infection and is characterized by tem...

Section: Atm Mediates P53 Induction During Mhv68 Replicationsupporting

confidence: 71%

Section: Atm Mediates P53 Induction During Mhv68 Replicationmentioning

confidence: 99%

Murine Gammaherpesvirus 68 LANA and SOX Homologs Counteract ATM-Driven p53 Activity during Lytic Viral Replication

Sifford

Stahl

Salinas

et al. 2016

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“…Levels of the tegument protein ORF75C delivered to infected BMDMs were similar between WT and ORF50stop virus at 4 hpi, prior to immediate-early gene expression by MHV68 (Fig. 6B, left) (66), and demonstrate comparable levels of particle delivery. By 9 hpi, there were slightly increased levels of ORF75C in WT-infected BMDMs compared to ORF50stop-infected cells (Fig.…”

Section: Resultsmentioning

confidence: 72%

Murine Gammaherpesvirus 68 Pathogenesis Is Independent of Caspase-1 and Caspase-11 in Mice and Impairs Interleukin-1β Production upon Extrinsic Stimulation in Culture

Cieniewicz

Dong

et al. 2015

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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-1␤ (IL-1␤) 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-1␤. In addition, MHV68 infection led to a reduction in IL-1␤ production after extrinsic lipopolysaccharide stimulation or upon coinfection with Salmonella enterica serovar Typhimurium. Interestingly, this impairment occurred at the proIL-1␤ transcript level and was independent of the RTA, the viral lytic replication and transcription activator. Taken together, MHV68 impairs the inflammasome response by inhibiting IL-1␤ production during the initial stages of infection. IMPORTANCEGammaherpesviruses 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-1␤ upon bacterial coinfection. Taken together, murine gammaherpesvirus impairment of the inflammatory cytokine IL-1␤ in macrophages identifies one mechanism by which the virus may inhibit caspase-1-dependent immune responses in the infected animal. G ammaherpesviruses establish lifelong latent infections that are associated with significant morbidity and the development of malignancies (1-3). Gammaherpesviruses initially infect the mucosal tissues of naive hosts and undergo productive replication prior to colonization of latency reservoirs, including B lymphocytes and macrophages (4-6). Murine gammaherpes...

mentioning

confidence: 99%

“…LANA is transcribed with immediate early kinetics upon G2HV infection of host cells, which suggests a role in productive viral replication (26,27). Indeed, LANA expression is robust throughout both the KSHV and MHV68 lytic replication cycles (26,(28)(29)(30)(31)(32). During MHV68 lytic infection, mLANA regulates viral gene expression, prevents premature cell death, and ultimately is required for efficient viral replication both in culture and in vivo (15,28,33,34).…”

mentioning

confidence: 99%

Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

Salinas

Byrum

Moreland

et al. 2016

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Latency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through coimmunoprecipitation and in vitro glutathione S-transferase (GST) pulldown assays. Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Pharmacologic inhibition and small hairpin RNA (shRNA)-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than that of WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc70 in facilitating MHV68 lytic replication. IMPORTANCELatency-associated nuclear antigen (LANA) is a conserved gamma-2-herpesvirus protein important for latency maintenance and pathogenesis. For MHV68, this includes regulating lytic replication and reactivation. While previous studies of KSHV LANA defined interactions with host cell proteins that impact latency, interactions that facilitate productive viral replication are not known. Thus, we performed a differential proteomics analysis to identify and prioritize cellular and viral proteins that interact with the MHV68 LANA homolog during lytic infection. Among the proteins identified was heat shock cognate protein 70 (Hsc70), which we determined is recruited to host cell nuclei in an mLANA-dependent process. Moreover, Hsc70 facilitates MHV68 protein expression and DNA replication, thus contributing to efficient MHV68 lytic replication. These experiments expand the known LANA-binding proteins to include MHV68 lytic replication and demonstrate a previously unappreciated role for Hsc70 in regulating viral...