Vera L. Tarakanova

Murine Gammaherpesvirus 68 Infection Is Associated with Lymphoproliferative Disease and Lymphoma in BALB β2 Microglobulin-Deficient Mice

ABSTRACT Human gammaherpesvirus infections are associated with development of lymphoproliferative disease. Understanding of the mechanisms of gammaherpesvirus lymphomagenesis during chronic infection in a natural host has been limited by the exquisite species specificity of human gammaherpesviruses and the expense of primates. Murine gammaherpesvirus γHV68 is genetically and biologically related to human gammaherpesviruses and herpesvirus saimiri and has been reported to be associated with lymphoproliferative disease in mice (N. P. Sunil-Chandra, J. Arno, J. Fazakerley, and A. A. Nash, Am. J. Pathol. 145:818-826, 1994). We report the development of an animal model of γHV68 lymphomagenesis in BALB/c β2 microglobulin-deficient mice (BALB β2m−/−). γHV68 infection induced two lymphoproliferative lesions: B-cell lymphoma and atypical lymphoid hyperplasia (ALH). ALH lesion histology resembled lesions of Epstein-Barr virus-associated posttransplant lymphoproliferative disease and was characterized by the abnormal infiltration of the white pulp with cells expressing the plasma cell marker CD138. Lymphomas observed in γHV68-infected animals were B220+/CD3− large-cell lymphomas. γHV68-infected cells were common in ALH lesions as measured by in situ hybridization with a probe specific for viral tRNAs (vtRNAs), but they were scarce in γHV68-infected spleens with normal histology. Unlike ALH lesions, γHV68 vtRNA-positive cells were rare in lymphomas. γHV68 infection of BALB β2m−/− mice results in lymphoproliferation and lymphoma, providing a valuable tool for identifying viral and host genes involved in gammaherpesvirus-associated malignancies. Our findings suggest that γHV68 induces lymphomas via hit-and-run oncogenesis, paracrine effects, or stimulation of chronic inflammation.

Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein

We have described three potential adenovirus type 5 (Ad5)-based replication-competent cancer gene therapy vectors named KD1, KD3, and VRX-007. All three vectors overexpress an Ad5 protein named Adenovirus Death Protein (ADP, also named E3–11.6 K protein). ADP is required for efficient lysis of Ad5-infected cells and spread of virus from cell to cell, and thus its overexpression increases the oncolytic activity of the vectors. KD1 and KD3 contain mutations in the Ad5 E1A gene that knock out binding of the E1A proteins to cellular p300/CBP and pRB; these mutations allow KD1 and KD3 to grow well in cancer cells but not in normal cells. VRX-007 has wild-type E1A. Here we report that radiation increases the oncolytic activity of KD1, KD3, and VRX-007. This increased activity was observed in cultured cells, and it was not because of radiation-induced replication of the vectors. The combination of radiation plus KD3 suppressed the growth of A549 lung adenocarcinoma xenografts in nude mice more efficiently than radiation alone or KD3 alone. The combination of ADP-overexpressing vectors and radiation may have potential in treating cancer.

Conserved gammaherpesvirus kinase and histone variant H2AX facilitate gammaherpesvirus latency in vivo

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus.

Murine Gammaherpesvirus 68 Genes both Induce and Suppress Lymphoproliferative Disease

ABSTRACT Gammaherpesvirus infection is associated with an increased incidence of lymphoproliferative disease in immunocompromised hosts. Murine gammaherpesvirus 68 (γHV68) infection of BALB β2-microglobulin-deficient (BALB β2m−/−) mice provides an animal model for analysis of the mechanisms responsible for the induction of a lymphoproliferative disease, atypical lymphoid hyperplasia (ALH), that is pathologically similar to posttransplant lymphoproliferative disease associated with Epstein-Barr virus infection. Here we report that the γHV68 v-cyclin and v-bcl-2 genes are required for the efficient induction of γHV68-associated ALH in BALB β2m−/− mice, while the v-GPCR gene is dispensable for ALH induction. In contrast to these findings, deletion of the viral M1 gene enhanced ALH. Thus, γHV68 genes can either inhibit or enhance the induction of lymphoproliferative disease in immunocompromised mice.

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.

Hsp90 inhibitor 17-DMAG decreases expression of conserved herpesvirus protein kinases and reduces virus production in Epstein-Barr virus-infected cells

ABSTRACT All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.

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.

Nitric Oxide Induces Ataxia Telangiectasia Mutated (ATM) Protein-dependent γH2AX Protein Formation in Pancreatic β Cells

Background: The mechanisms that control β cell fate following cytokine- and nitric oxide-induced damage remain unknown. Results: Cytokine-induced nitric oxide activates ATM and ATM-dependent caspase activation in β cells. Conclusion: ATM regulates the induction of apoptosis in cytokine-treated β cells. Significance: These studies define a role for DNA damage and ATM activation in nitric oxide-induced β cell apoptosis. In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

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.