Functional role of PACT in limiting influenza A virus replication

Abstract

Innate immunity is the first line of defense against pathogen invasion. A group of pathogen sensors known as pattern recognition receptors (PRRs) are present in human cells for efficient sensing of potential threats. These PRRs are able to discriminate non-self materials through physical association with components uniquely possessed by pathogens. In the context of infection by RNA viruses, RIG-I-like receptors (RLRs) are the main PRRs responsible for sensing intracellular viral RNA (vRNA). Upon ligand binding, RLRs activate a cascade of signaling that eventually leads to production of type I interferons (IFN-Is), the cytokines that serve as danger signals. IFN-Is stimulate cells to transcribe a subset of antiviral genes called interferon stimulated genes (ISGs) which collectively squelch viral infection. Besides RLRs, some double-stranded RNA binding proteins (dsRBPs) including PKR, OAS and ADAR1 are also involved in antiviral immunity independent of interferon induction. Our group recently identified a dsRBP called PACT that activates RLRs and promotes optimal RLR-mediated interferon production. However, the dsRBP nature of PACT also prompted us to evaluate the potential interferon-independent antiviral role of PACT. In this study, influenza A virus (IAV) infection was used as a model to demonstrate the functionality of PACT. Using in vitro assay, I have shown that PACT directly bound IAV RNA derived from infected cells. Deep sequencing data suggested PACT’s specific discrimination of viral RNA from the host. RNA derived from all 8 segments of IAV could be recognized by PACT, and the RNA bound were likely double-stranded in nature. The PACT:vRNA binding can also be reproduced in infected cells by RNA-IP assay. Moreover, using our PACT knockout infection model, we demonstrated the ability of PACT to inhibit viral replication independently of interferon signaling. The loss of PACT also caused attenuated viral control as revealed by increased viral titer. Taken together, I demonstrated a previously undescribed interferon-independent antiviral role of PACT. In light of the effectiveness of innate immunity in combating viral infection, many viruses have evolved to encode viral proteins known as interferon antagonists to subvert host interferon response. The Human T-cell lymphotropic virus type I (HTLV-1) possesses an interferon antagonist Tax that blunts ISG production upon induction by IFN-I. However, no viral proteins of HTLV-1 have been reported to inhibit IFN-I production. Here we postulated that Tax might play such role in addition to its known suppression of ISG production, given the fact that many viral proteins are multifaceted and Tax is the only reported interferon antagonist in HTLV-1. In this study, I have shown that HTLV-1-infected cells were incapable of producing IFN-Is. Expression of Tax alone was enough to account for this suppression and this function of Tax is independent of its CREB- and NFκB-activating ability. Mechanistic studies showed that Tax affected phosphorylation of IRF3, the transcription factor required for IFN-I production. Tax was further shown to bind TBK1 and inhibit its kinase activity, leading to diminished IRF3 phosphorylation and so IFN-I production. Collectively, I reported the ability of HTLV-1 Tax to suppress IFN-I production through inhibition of TBK1.