Research Article: Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2

Date Published: December 1, 2016

Publisher: Public Library of Science

Author(s): Jennifer H. Stuart, Rebecca P. Sumner, Yongxu Lu, Joseph S. Snowden, Geoffrey L. Smith, Yan Xiang.


The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

Partial Text

The innate immune response is the first line of defense against invading pathogens. Interferons (IFNs) are one of the key players in this early response to infection and are particularly important to protect against viruses, as can be seen by the increased susceptibility of IFNα/β receptor (IFNAR) knock out mice to viral infections [1]. There are two main branches to the IFN response; their production and the signalling initiated in response to the binding of secreted IFNs to their receptors at the cell surface.

Previously, C6 was identified as a VACV immunomodulator and virulence factor and was shown to inhibit the induction of type I IFNs through inhibition of the IRF-3/7 signalling pathway at the level of the TBK1/IKKε kinase complex [26]. This study identifies a second function for VACV protein C6 as an inhibitor of the cellular response to type I IFN. Data presented demonstrate that C6 inhibits IFNα-induced expression of ISGs at both the mRNA and protein level (Fig 1). The inability of a pharmacological inhibitor of TBK1/IKKε to reduce IFNα-induced reporter gene expression (S2 Fig) indicates that inhibition of this kinase complex by C6 is unlikely to explain the ability of C6 to also inhibit the cellular response to IFNα. Therefore, to elucidate the mechanism by which C6 has its inhibitory effect on this second pathway, the IFNα-induced phosphorylation and nuclear translocation of STAT1 and STAT2 were examined and C6 was found to have no effect on these early events of this signalling pathway (Figs 2, 4 and 5). Furthermore, both endogenous STAT2 and a constitutively active ISGF3 mimic, IRF-9-S2C, were still able to bind to the ISRE in the presence of C6 (Fig 7), indicating C6 exerts its inhibitory effect after ISGF3 binding to the ISRE. Interestingly, C6 interacts with STAT2 (Fig 8A–8C) and the transactivation domain (aa 747–851) of STAT2 fused to IRF-9 (Fig 8D) but not with STAT1 or IRF-9 (Fig 8A). The STAT2 transactivation domain is known to be required for the recruitment of chromatin modifiers and transcriptional machinery [37]. Therefore, the ability of C6 to interact specifically with this domain gives insight into how this viral protein may inhibit this crucial signalling pathway at such a late stage.




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