In this study, we demonstrated that HCV core protein plays an important role in subverting host innate immunity by blocking type I IFN signaling. We further showed that HCV core blocks Jak-STAT signaling by direct physical interaction with STAT1. The HCV core-STAT1 interaction maps to the N-terminal 23 amino acids of HCV core and the SH2 region of STAT1 (aa 578 to 684). These interactions are required for the observed loss of P-STAT1 and impairment of IFN signaling.
The type I IFN system is the first line of host defense against virus infection. HCV, like other viruses, has evolved a unique strategy to disrupt host IFN-induced innate antiviral responses through the action of its core protein. HCV core has protean actions. It is capable of broadly signaling across multiple transduction pathways that result in pleiotropic effects in suppressing host immunity and enhancing HCV replication (22
). Among the best characterized of these pathways in hepatocyte-derived lines are the MAPK/extracellular signal-regulated kinase (13
) and NF-κB pathways (18
). The HCV core N terminus (aa 1 to 20) has been demonstrated to bind to HCV IRES RNA and inhibit HCV IRES-dependent translation (22
). There is also evidence that HCV core acts to suppress type I IFN signaling at several levels (3
). For instance, HCV core mediates the disruption of STAT1 phosphorylation through the induction of SOCS3 (5
). It can also inhibit P-STAT1 nuclear import (4
). In this study, we found that the expression of the major Jak-STAT functional component P-STAT1 was markedly reduced in HCV-core-transfected cells. C-terminally truncated but not N-terminally truncated HCV core constructs produced parallel reductions in STAT1 binding, P-STAT1 loss, and blockage of IFN signaling. The HCV core binding and functional activities specifically map to its 23 N-terminal amino acids.
We further demonstrated that the STAT1 SH2 domain appears to be the critical HCV core interaction domain. The STAT1 protein sequence can be divided into N-terminal and C-terminal regions. The STAT1 protein N-terminal region includes the NH2 region (aa 1 to 35), the coiled-coil domain (aa 135 to 315), and the DNA binding domain (315 to 487), while the C-terminal region includes the SH3 domain (aa 487 to 576), SH2 domain (aa 576 to 683), and transactivation domain (aa 683 to 750) (8
) (Fig. ). The STAT1 SH2 domain functions as a recognition site for phosphorylation by JAK kinases (7
). The STAT1 SH2 domain is critical for two functions: (i) the recruitment of STAT1 to JAK kinases and its subsequent phosphorylation by JAK kinase and (ii) the promotion of homo- or heterodimerization of STAT1. STAT1 SH2 heterodimerization with STAT2 and binding to interferon regulatory factor 9 are essential for the formation of a heterodimerization DNA binding complex that induces ISRE-mediated transcription of ISGs (8
In this study, we have shown that the interaction between HCV core and STAT1 localizes to the STAT1 SH2 region (aa 576 to 683) and that N-terminal HCV core mutants that fail to bind STAT1 do not block P-STAT1 formation. A plausible model explaining the observed events may proceed as follows: the binding of HCV core to the STAT1 SH2 domain inhibits the recruitment of STAT1 to JAK kinases, in turn inhibiting JAK-induced phosphorylation of STAT1. In addition, STAT1-STAT2 heterodimerization may be inhibited by both blockade of the SH2 domain and inhibition of STAT1 phosphorylation. The net result of these events is decreased DNA binding of ISGs. Our previous finding demonstrating that HCV expression is associated with reduced ISGF3 formation is consistent with this model (23
). Our data also show that point mutations at other key STAT1 regulatory sites do not disrupt the HCV core-STAT1 interaction, implying that these regulatory sites are not blocked by HCV core.
STAT1 deletion studies have demonstrated that its N terminus is required for its proteasome-mediated degradation by mumps protein V (40
). Rabies virus P protein has been reported to interact with STAT1 and blocks the IFN signaling pathway (37
). The carboxy-terminal portion of rabies virus P protein has been demonstrated to inhibit the IFN signal transduction pathway by interacting with a region containing the STAT1 DNA binding domain (aa 315 to 487) and the coiled-coil domain (aa 135 to 315) (37
). On the other hand, C-terminal STAT1 (aa 577 to 750) was reported to be unnecessary for mumps virus V protein-induced STAT1 degradation (40
). There are several reports of blocked IFN signaling caused by the degradation of STAT1 induced by V proteins encoded by human parainfluenza virus type 2 and rubulaviruses simian virus 5, simian virus 41, mumps virus, and Newcastle disease virus (28
). These viruses have each evolved different mechanisms to counteract STAT1-driven type I IFN signaling. For HCV, we propose a unique model in which the interaction of the HCV core N-terminal domain (aa 1 to 23) with the STAT1 SH2 domain (aa 576 to 683) results in decreased STAT1 phosphorylation, leading to decreased STAT1-STAT2 heterodimerization, reduced ISGF3 formation, decreased DNA binding to the ISRE of IFN-stimulated genes, and, ultimately, decreased IFN-stimulated gene transcription. These findings have important implications for the understanding of complex interactions between HCV proteins and the host cell antiviral defenses and may lead to novel strategies to interrupt the HCV core-STAT1 interaction and its adverse downstream effects on IFN signal transduction. We speculate that HCV core, in addition to blocking the formation of P-STAT1, also promotes ubiquitin-mediated proteasome-dependent degradation of STAT1. Further studies to address whether HCV core directly promotes STAT1 degradation are warranted.