Viral and Cellular Genomes Activate Distinct DNA Damage Responses

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: Discussionmentioning

confidence: 99%

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

Sifford

Stahl

Salinas

et al. 2016

“…2A). In addition to modulating sumoylation, E1B-55K and E4-ORF3 have a critical role in inactivating MRN and ATM to facilitate viral genome replication (45,(59)(60)(61). We show that the lack of SUMO2-associated E2A domains in ⌬E1B-55K/⌬E4-ORF3-infected cells is due to an upstream MRN-ATM-mediated checkpoint that prevents viral genome replication.…”

Section: Discussionmentioning

confidence: 85%

“…E1B-55K or E4-ORF3 is required to inactivate a critical early MRN-ATM checkpoint to viral genome replication (45). Therefore, the lack of SUMO2 at E2A domains could be an indirect consequence of an upstream block in viral genome replication in ⌬E1B-55K/⌬E4-ORF3-infected cells.…”

Section: Resultsmentioning

confidence: 99%

“…Alternatively, it could reflect a direct role of E1B-55K/E4-ORF3 in regulating SUMO2 localization and organization at E2A domains. To distinguish between these possibilities, we used the ATM kinase inhibitor KU-55933 (46) to rescue viral genome replication in ⌬E1B-55K/⌬E4-ORF3-infected cells (45). We also analyzed ⌬E1B-55K/⌬E4-ORF3 superinfected cells (MOI of 250), as previous studies have shown that high MOIs can rescue the replication defect of E4-deleted viruses (47)(48)(49).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Adenovirus E4-ORF3 Targets PIAS3 and Together with E1B-55K Remodels SUMO Interactions in the Nucleus and at Virus Genome Replication Domains

Higginbotham

O’Shea

2015

Adenovirus E4-ORF3 and E1B-55K converge in subverting critical overlapping cellular pathways to facilitate virus replication. Here, we show that E1B-55K and E4-ORF3 induce sumoylation and the assembly of SUMO2/3 viral genome replication domains. Using a conjugation-deficient SUMO2 construct, we demonstrate that SUMO2/3 is recruited to E2A viral genome replication domains through noncovalent interactions. E1B-55K and E4-ORF3 have critical functions in inactivating MRN and ATM to facilitate viral genome replication. We show that ATM kinase inhibitors rescue ⌬E1B-55K/⌬E4-ORF3 viral genome replication and that the assembly of E2A domains recruits SUMO2/3 independently of E1B-55K and E4-ORF3. However, the morphology and organization of SUMO2/3-associated E2A domains is strikingly different from that in wild-type Ad5-infected cells. These data reveal that E1B-55K and E4-ORF3 specify the nuclear compartmentalization and structure of SUMO2/3-associated E2A domains, which could have important functions in viral replication. We show that E4-ORF3 specifically targets and sequesters the cellular E3 SUMO ligase PIAS3 but not PIAS1, PIAS2, or PIAS4. The assembly of E4-ORF3 into a multivalent nuclear matrix is required to target PIAS3. In contrast to MRN, PIAS3 is targeted by E4-ORF3 proteins from disparate adenovirus subgroups. Our studies reveal that PIAS3 is a novel and evolutionarily conserved target of E4-ORF3 in human adenovirus infections. Furthermore, we reveal that viral proteins not only disrupt but also usurp SUMO2/3 to transform the nucleus and assemble novel genomic domains that could facilitate pathological viral replication. IMPORTANCESUMO is a key posttranslational modification that modulates the function, localization, and assembly of protein complexes. In the ever-escalating host-pathogen arms race, viruses have evolved strategies to subvert sumoylation. Adenovirus is a small DNA tumor virus that is a global human pathogen and key biomedical agent in basic research and therapy. We show that adenovirus infection induces global changes in SUMO localization and conjugation. Using virus and SUMO mutants, we demonstrate that E1B-55K and E4-ORF3 disrupt and usurp SUMO2/3 interactions to transform the nucleus and assemble highly structured and compartmentalized viral genome domains. We reveal that the cellular E3 SUMO ligase PIAS3 is a novel and conserved target of E4-ORF3 proteins from disparate adenovirus subgroups. The induction of sumoylation and SUMO2/3 viral replication domains by early viral proteins could play an important role in determining the outcome of viral infection. V iruses usurp and trigger cellular signaling cascades that have dynamic and system-wide consequences for host-pathogen interactions. Protein posttranslational modifications have the power to alter the functions, structural interactions, and localization of cellular and viral proteins to determine the outcome of infection. For example, studies with polyomavirus middle T antigen and v-Src-associated kinase activities led to the dis...

“…ATM facilitates replication of diverse RNA and DNA viruses in cell culture. Although this proviral activity is traditionally ascribed to the role of ATM in DNA damage response (4,5), it is likely that regulation of additional signaling networks by ATM contributes to viral replication (6).…”

mentioning

confidence: 99%

B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection

Darrah

Kulinski

Mboko

et al. 2017

Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection, as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in a host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses in vitro, suggesting that ATM is proviral in the context of infected cell cultures. The proviral role of ATM is also evident in vivo, as myeloidspecific ATM expression facilitates MHV68 reactivation during the establishment of viral latency. In order to better understand the complex relationship between host ATM and gammaherpesvirus infection, we depleted ATM specifically in B cells, a cell type critical for chronic gammaherpesvirus infection. B cell-specific ATM deficiency attenuated the establishment of viral latency due to compromised differentiation of ATM-deficient B cells. Further, we found that during long-term infection, peritoneal B-1b, but not related B-1a, B cells display the highest frequency of gammaherpesvirus infection. While ATM expression did not affect gammaherpesvirus tropism for B-1 B cells, B cell-specific ATM expression was necessary to support viral reactivation from peritoneal cells during long-term infection. Thus, our study reveals a role of ATM as a host factor that promotes chronic gammaherpesvirus infection of B cells.IMPORTANCE Gammaherpesviruses infect a majority of the human population and are associated with cancer, including B cell lymphomas. ATM is a unique host kinase that has both proviral and antiviral roles in the context of gammaherpesvirus infection. Further, there is insufficient understanding of the interplay of these roles in vivo during chronic infection. In this study, we show that ATM expression by splenic B cells is required for efficient establishment of gammaherpesvirus latency. We also show that ATM expression by peritoneal B cells is required to facilitate viral reactivation during long-term infection. Thus, our study defines a proviral role of B cellspecific ATM expression during chronic gammaherpesvirus infection.KEYWORDS ATM, gammaherpesvirus, B cell, T cell response, reactivation, chronic infection A taxia-telangiectasia (A-T) mutated (ATM) kinase is a multifunctional kinase, insufficiency of which in humans manifests as A-T (1, 2). The function of ATM was originally defined in the context of response to double-stranded DNA breaks. However,