Bryan C. Mounce

Interferon regulatory factor-1 restricts gammaherpesvirus replication in primary immune cells.

ABSTRACT Gammaherpesviruses are ubiquitous pathogens that establish a lifelong infection and are associated with cancer. In spite of the high seroprevalence of infection, the risk factors that predispose the host toward gammaherpesvirus-induced malignancies are still poorly understood. Interferon (IFN) regulatory factor 1 (IRF-1) is a tumor suppressor that is also involved in the regulation of innate and adaptive immune responses. On the basis of its biology, IRF-1 represents a plausible host factor to attenuate gammaherpesvirus infection and tumorigenesis. In this study, we show that IRF-1 restricts gammaherpesvirus replication in primary macrophages, a physiologically relevant immune cell type. In spite of the known role of IRF-1 in stimulating type I IFN expression, induction of a global type I IFN response was similar in IRF-1-deficient and -proficient macrophages during gammaherpesvirus infection. However, IRF-1 was required for optimal expression of cholesterol-25-hydroxylase, a host enzyme that restricted gammaherpesvirus replication in primary macrophages and contributed to the antiviral effects of IRF-1. In summary, the current study provides an insight into the mechanism by which IRF-1 attenuates gammaherpesvirus replication in primary immune cells, a mechanism that is likely to contribute to the antiviral effects of IRF-1 in other virus systems. IMPORTANCE Interferon regulatory factor 1 (IRF-1) is a transcription factor that regulates innate and adaptive immune responses and functions as a tumor suppressor. IRF-1 restricts the replication of diverse viruses; however, the mechanisms responsible for the antiviral effects of IRF-1 are still poorly understood. Gammaherpesviruses are ubiquitous pathogens that are associated with the induction of several malignancies. Here we show that IRF-1 expression attenuates gammaherpesvirus replication in primary macrophages, in part by increasing expression of cholesterol-25-hydroxylase (CH25H). CH25H and its product, 25-hydroxycholesterol, restrict replication of diverse virus families. Thus, our findings offer an insight into the mechanism by which IRF-1 attenuates the replication of gammaherpesviruses, a mechanism that is likely to be applicable to other virus systems.

Ataxia telangiectasia mutated kinase controls chronic gammaherpesvirus infection.

ABSTRACT Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.

Coordinate Regulation of DNA Damage and Type I Interferon Responses Imposes an Antiviral State That Attenuates Mouse Gammaherpesvirus Type 68 Replication in Primary Macrophages

ABSTRACT DNA damage response (DDR) is a sophisticated cellular network that detects and repairs DNA breaks. Viruses are known to activate the DDR and usurp certain DDR components to facilitate replication. Intriguingly, viruses also inhibit several DDR proteins, suggesting that this cellular network has both proviral and antiviral features, with the nature of the latter still poorly understood. In this study we show that irradiation of primary murine macrophages was associated with enhanced expression of several antiviral interferon (IFN)-stimulated genes (ISGs). ISG induction in irradiated macrophages was dependent on type I IFN signaling, a functional DNA damage sensor complex, and ataxia-telangiectasia mutated kinase. Furthermore, IFN regulatory factor 1 was also required for the optimal expression of antiviral ISGs in irradiated macrophages. Importantly, DDR-mediated activation of type I IFN signaling contributed to increased resistance to mouse gammaherpesvirus 68 replication, suggesting that the coordinate regulation of DDR and type I IFN signaling may have evolved as a component of the innate immune response to virus infections.

Dynamic association of gammaherpesvirus DNA with core histone during de novo lytic infection of primary cells

Association of herpesvirus DNA with histones has important implications for lytic and latent infections; thus herpesviruses arbitrate interactions with histones to productively infect host cells. While regulation of alpha and betaherpesvirus chromatin during lytic infection has been actively investigated, very little is known about interaction of gammaherpesvirus DNA with histones upon de novo lytic infection. Murine gammaherpesvirus-68 (MHV68) is a rodent pathogen that offers a tractable system to study gammaherpesvirus lytic infection in primary cells. In this study we report that MHV68 promoter and orilyt sequences underwent dynamic association with histone H3 during de novo lytic infection of primary macrophages and fibroblasts. Similar to HSV-1, the degree of MHV68 DNA association with histone H3 was dependent on the multiplicity of infection and was further regulated by viral DNA synthesis. Our work sets a precedent for future studies of gammaherpesvirus chromatin during de novo lytic infection.

Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

Mouse gammaherpesvirus-68 infection acts as a rheostat to set the level of type I interferon signaling in primary macrophages

Type I interferon (IFN) is a critical antiviral response of the host. We found that Interferon Regulatory Factor 3 (IRF-3) was responsible for induction of type I IFN following mouse gammaherpesvirus-68 (MHV68) infection of primary macrophages. Intriguingly, type I IFN signaling was maintained throughout the entire MHV68 replication cycle, in spite of several known viral IFN antagonists. However, MHV68-infected primary macrophages displayed attenuated responses to exogenous type I IFN, suggesting that MHV68 controls the level of type I IFN signaling that is allowed to occur during replication. Type I IFN receptor and IRF-3 were necessary to attenuate transcription of MHV68 RTA, an immediate early gene critical for replication. Furthermore, higher constitutive activity of RTA promoters was observed in the absence of type I IFN signaling. Our study suggests that MHV68 has preserved the ability to sense type I IFN status of the host in order to limit lytic replication.

A Conserved Gammaherpesvirus Protein Kinase Targets Histone Deacetylases 1 and 2 To Facilitate Viral Replication in Primary Macrophages

ABSTRACT Gammaherpesviruses are ubiquitious pathogens that establish lifelong infection and are associated with several malignancies. All gammaherpesviruses encode a conserved protein kinase that facilitates viral replication and chronic infection and thus represents an attractive therapeutic target. In this study, we identify a novel function of gammaherpesvirus protein kinase as a regulator of class I histone deacetylases (HDAC). Mouse gammaherpesvirus 68 (MHV68)-encoded protein kinase orf36 interacted with HDAC1 and 2 and prevented association of these HDACs with the viral promoter driving expression of RTA, a critical immediate early transcriptional activator. Furthermore, the ability to interact with HDAC1 and 2 was not limited to the MHV68 orf36, as BGLF4, a related viral protein kinase encoded by Epstein-Barr virus, interacted with HDAC1 in vitro. Importantly, targeting of HDAC1 and 2 by orf36 was independent of the kinases enzymatic activity. Additionally, orf36 expression, but not its enzymatic activity, induced changes in the global deacetylase activity observed in infected primary macrophages. Combined deficiency of HDAC1 and 2 rescued attenuated replication and viral DNA synthesis of the orf36 null MHV68 mutant, indicating that the regulation of HDAC1 and 2 by orf36 was relevant for viral replication. Understanding the mechanism by which orf36 facilitates viral replication, including through HDAC targeting, will facilitate the development of improved therapeutics against gammaherpesvirus kinases.

Primary Macrophages Rely on Histone Deacetylase 1 and 2 Expression To Induce Type I Interferon in Response to Gammaherpesvirus Infection

ABSTRACT Type I interferon is induced shortly following viral infection and represents a first line of host defense against a majority of viral pathogens. Not surprisingly, both replication and latency of gammaherpesviruses, ubiquitous cancer-associated pathogens, are attenuated by type I interferon, although the mechanism of attenuation remains poorly characterized. Gammaherpesviruses also target histone deacetylases (HDACs), a family of pleiotropic enzymes that modify gene expression and several cell signaling pathways. Specifically, we have previously shown that a conserved gammaherpesvirus protein kinase interacts with HDAC1 and -2 to promote gammaherpesvirus replication in primary macrophages. In the current study, we have used genetic approaches to show that expression of HDAC1 and -2 is critical for induction of a type I interferon response following gammaherpesvirus infection of primary macrophages. Specifically, expression of HDAC1 and -2 was required for phosphorylation of interferon regulatory factor 3 (IRF3) and accumulation of IRF3 at the beta interferon promoter in gammaherpesvirus-infected primary macrophages. To our knowledge, this is the first demonstration of a specific role for HDAC1 and -2 in the induction of type I interferon responses in primary immune cells following virus infection. Furthermore, because HDAC1 and -2 are overexpressed in several types of cancer, our findings illuminate potential side effects of HDAC1- and -2-specific inhibitors that are currently under development as cancer therapy agents. IMPORTANCE Gammaherpesviruses establish chronic infection in a majority of the adult population and are associated with several malignancies. Infected cells counteract gammaherpesvirus infection via innate immune signaling mediated primarily through type I interferon. The induction of type I interferon expression proceeds through several stages using molecular mechanisms that are still incompletely characterized. In this study, we show that expression of HDAC1 and -2 by macrophages is required to mount a type I interferon response to incoming gammaherpesvirus. The involvement of HDAC1 and -2 in the type I interferon response highlights the pleiotropic roles of these enzymes in cellular signaling. Interestingly, HDAC1 and -2 are deregulated in cancer and are attractive targets of new cancer therapies. Due to the ubiquitous and chronic nature of gammaherpesvirus infection, the role of HDAC1 and -2 in the induction of type I interferon responses should be considered during the clinical development of HDAC1- and -2-specific inhibitors.

ATM facilitates mouse gammaherpesvirus reactivation from myeloid cells during chronic infection

Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo.

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

ABSTRACT 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 replication and transcription activator. 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.