Influenza A virus NS1 protein targets double stranded RNA-binding protein PACT to suppress innate antiviral response

Abstract

BACKGROUND: Influenza A virus (IAV) is a common human pathogen causing devastating pandemics and seasonal epidemics. Innate antiviral immune response exemplified by the production of type I interferons (IFNs) is induced when IAV is recognized by Toll-like receptors and RIG-I-like receptors. To ensure its successful infection and replication, IAV encodes IFN-antagonizing non-structural protein NS1, a multi-functional double-stranded RNA binding protein that interacts with both RIG-I and PKR to circumvent host antiviral defense. RIG-I is a cytosolic sensor of IAV. We have previously shown that RIG-I requires PACT for full activity. PACT is a cellular dsRNA-binding protein originally identified to be an activator of PKR. How IAV NS1 might affect the function of PACT in innate antiviral response remains to be understood. METHODS AND RESULTS: We demonstrated that NS1 physically interacts with PACT to perturb RIG-I-dependent IFN production. We also characterized the role of PACT in NS1-mediated suppression of RIG-I-induced IFN response. PACT-mediated potentiation of RIG-I activation on IFN-ß reporter activity was significantly inhibited by NS1. PACT also acts as an inhibitor of IAV polymerase transcriptional activity. By RNA-nucleoprotein complex (RNP) reconstitution assay, it was shown that overexpression of PACT could significantly dampen the RNP activity. The observed PACT-mediated suppression of RNP activity could be partially rescued by overexpression of NS1 protein. We also observed that PACT-knockdown in cells promoted IAV replication. Furthermore, we demonstrated IFN independence of PACT-mediated suppression of RNP activity. CONCLUSION AND IMPLICATIONS: Our results show that PACT is a novel target of IAV IFN-antagonizing protein NS1 and suggest the counteracting roles of PACT and NS1 during IAV infection. We also provide evidence on the IFN-independent effector function for PACT on counteracting IAV replication. Our findings provide new insights into the inhibitory mechanism of PACT on IAV replication. Small molecule agonists of PACT might be used as adjuvants and anti-influenza agents. Thus, our work also reveals new strategies for development of anti-influenza drugs. ACKNOWLEDGEMENT: Supported by RGC (HKU1/CRF/11G and N-HKU712/12), HMRF (12111312, 14130862 and CID-III) and S. K. Yee MRF (2011).