Search

Unlabelled: 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., Importance: Gammaherpesviruses 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: 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., Importance: Latency-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 replication., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)