Murine Gammaherpesvirus 68 Encoding Open Reading Frame 11 Targets TANK Binding Kinase 1 To Negatively Regulate the Host Type I Interferon Response

Abstract

Upon viral infection, type I interferons, such as alpha and beta interferon (IFN-␣ and IFN-␤, respectively), are rapidly induced and activate multiple antiviral genes, thereby serving as the first line of host defense. Many DNA and RNA viruses counteract the host interferon system by modulating the production of IFNs. In this study, we report that murine gammaherpesvirus 68 (MHV-68), a double-stranded DNA virus, encodes open reading frame 11 (ORF11), a novel immune modulator, to block IFN-␤ production. ORF11-deficient recombinant viruses induced more IFN-␤ production in fibroblast and macrophage cells than the MHV-68 wild type or a marker rescue virus. MHV-68 ORF11 decreased IFN-␤ promoter activation by various factors, the signaling of which converges on TBK1-IRF3 activation. MHV-68 ORF11 directly interacted with both overexpressed and endogenous TBK1 but not with IRF3. Physical interactions between ORF11 and endogenous TBK1 were further confirmed during virus replication in fibroblasts using a recombinant virus expressing FLAG-ORF11. ORF11 efficiently reduced interaction between TBK1 and IRF3 and subsequently inhibited activation of IRF3, thereby negatively regulating IFN-␤ production. Our domain-mapping study showed that the central domain of ORF11 was responsible for both TBK1 binding and inhibition of IFN-␤ induction, while the kinase domain of TBK1 was sufficient for ORF11 binding. Taken together, these results suggest a mechanism underlying inhibition of IFN-␤ production by a gammaherpesvirus and highlight the importance of TBK1 in DNA virus replication. IMPORTANCEGammaherpesviruses are important human pathogens, as they are associated with various kinds of tumors. Upon virus infection, the type I interferon pathway is activated by a series of signaling molecules and stimulates antiviral gene expression. To subvert such interferon antiviral responses, viruses are equipped with multiple factors that can inhibit its critical steps. In this study, we took an unbiased genomic approach using a mutant library of murine gammaherpesvirus 68 to screen a novel viral immune modulator that negatively regulates the type I interferon pathway and identified ORF11 as a strong candidate. ORF11-deficient virus infection produced more interferon than the wild type in both fibroblasts and macrophages. During virus replication, ORF11 directly bound to TBK1, a key regulatory protein in the interferon pathway, and inhibited TBK1-mediated interferon production. Our results highlight a crucial role of TBK1 in controlling DNA virus infection and a viral strategy to curtail host surveillance.

Downregulation of Poly(ADP-Ribose) Polymerase 1 by a Viral Processivity Factor Facilitates Lytic Replication of Gammaherpesvirus

“…KSHV ORF59 encodes PF-8, which is essential for virus genome replication (13)(14)(15)25) as an early late protein (26,27). To test whether these PARP-1-interacting proteins may induce PARP-1 downregulation, we transiently overexpressed KSHV RTA (22), ORF49, and PF-8 in HEK293T cells (19). The results showed that endogenous PARP-1 was downregulated in cells transfected with PF-8 but not in cells transfected with RTA or ORF49 ( Fig.…”

“…Polyethylenimine (1 mg/ml; Sigma) was used for the transfection of HEK293T cells, as previously described (19). To obtain PF-8-expressing lentiviruses, a FLAG-tagged PF-8-containing lentiviral vector and packaging vector (kind gifts from Seungmin Hwang, The University of Chicago, Chicago, IL, USA) were cotransfected into HEK293T cells.…”

“…HEK293T cells were seeded and transfected with the plasmids indicated above for 48 h. BC-3 cells or HEK293T cells expressing .4], 100 mM NaCl, 0.5% NP-40, and 1% Triton X-100) containing 1/100 volume of a protease inhibitor cocktail (Sigma). Immunoprecipitation was performed as previously described (19), and the precipitated proteins were analyzed by Western blotting. Immunofluorescence assay.…”

Interplay between Kaposi's sarcoma-associated herpesvirus and the innate immune system

“…For example, vIL6 prevents the formation of the ISGF3 complex possibly by blocking Tyk2 phosphorylation [153] while vIRF2 inhibits ISGF3-mediated transcription [212]. In addition, functional screens in of protein-coding genes of KSHV, EBV and MHV-68 have identified a handful of distantly related IFN antagonists [213-216] referred to as dUTPase-related proteins (DURPs), which comprise ORF10, ORF11 and ORF54 [217]. ORF10 was shown to form an inhibitory complex that includes IFNAR subunits, Jak1 and Tyk2, and STAT2 [214].…”

“…While the anti-IFN effect of KSHV ORF54 has not been mechanistically characterized, its MHV-68 homologue induces degradation of IFNAR1 [215]. Currently, there is no report regarding KSHV ORF11 and its potential involvement in IFN signaling but it has been shown that the MHV-68 and EBV homologues can antagonize the type I IFN response, albeit via distinct mechanisms [213, 216]. Thus, DURPs appear to be an emerging family of anti-IFN genes analogous to the vIRFs.…”