Study of functions of mammalian argonaute 2 in inhibiting RIG-I-mediated antiviral signaling pathway and interference of influenza A virus replication

Abstract

Influenza A viruses (IAVs) have a significant impact on public health worldwide, causing annual seasonal epidemics and occasional pandemics. In response to infection with RNA viruses, including influenza A virus, the host antiviral innate immune response is effectively triggered through recognition of viral RNA by the receptor RIG-I. However, the molecular mechanisms through which RIG-I function is modulated are not completely understood. In this thesis, I utilized influenza A virus as an infection model to study the functions of mammalian Argonaute 2 (Ago2) in RIG-I-mediated antiviral signaling and influenza A virus replication. My results showed that Ago2 knockout significantly increased the phosphorylation of IRF3 and Stat1, key steps in RIG-I-mediated signaling, and also transcription of a subset of antiviral genes, following virus infection. Meanwhile, restoration of Ago2 by ectopic expression reversed the enhancement of IRF3 and Stat1 phosphorylation and activation of genes downstream of RIG-I signaling in Ago2 knockout cells. In addition, functional analyses using various technologies clearly demonstrated that negative regulation of RIG- I antiviral responses in the early stages of virus infection was Ago2 specific and independent of canonical miRNA or siRNA pathways. I further showed that Ago2 was able to bind to both viral genomic RNAs and mRNAs and that expression of IFNβ was induced only by RIG-I agonists, and not by downstream effectors, in Ago2-deficient cells. These findings suggest that Ago2 regulation is functional in the early steps of RIG-I-mediated receptor pathways. To characterize the molecular basis for Ago2-mediated negative regulation of RIG- I-triggered antiviral innate immune responses, RNA immunoprecipitation (RIP) assays were performed, confirming that Ago2 interacted with 5’ppp viral RNAs directly. More importantly, mechanistic studies revealed that Ago2 interacted strongly with 2-Card and the helicase domain of RIG-I and competed for binding of viral 5’ppp RNAs with RIG-I, thereby inhibiting activation of the RIG-I-MA VS signaling pathway. Further characterization indicated that Ago2 also interfered with the process of RIG-I filament formation and MAVS activation. Aiming to understand the role of Ago2 in virus replication, my study found that Ago2 inhibited influenza A virus replication in both early and late stages of infection. Notably, inhibition of virus replication and suppression of RIG-I-mediated signaling pathways by Ago2 were mutually independent. My study further showed that inhibition of virus in the early hours of infection was likely caused by Ago2 regulation at the post-transcriptional level while the complex effects, of which included miRNAs direct regulation of the immune response, miRNAs indirect regulation of virus replication and specific inhibition of viral proteins and RNAs exerted by Ago2, affected virus replication at late time-point infection. In summary, my study identified a novel mechanism by which Ago2 negatively regulated RIG-I-mediated antiviral signaling pathways through competing with RIG-I for viral RNA binding and interfering with the process of RIG-I filament formation and MAVS activation. I also discovered that Ago2 served as a host restriction factor, suppressing virus replication during both early and late stages of infection.