Function of mammalian Argonaute 2 in influenza A virus infection

Abstract

Argonaute 2 protein, namely AGO2, is a key component of RNA-induced silencing complex (RISC) that regulates cellular processes on post-transcriptional levels by miRNA and siRNA silencing. Although it is still unclear whether miRNA and siRNA play a significant role in early phase of influenza A virus (IAV) infection (1-4), we observed that depletion of AGO2 resulted in dramatic increase in IAV replication in virus infected cells. Therefore, we herein aim to explore the functions of Argonaute 2 in IAV infection. In this study, we used wildtype and delNS1 mutant WSN viruses as a working model. We also generated Ago2-KO cell lines (A549, 293, and 293T) using CRISPR/Case9 technology. At early time-point of infection, expression of viral proteins (in particular, HA, NS1, M1 and M2 proteins) increased significantly in infected AGO2-KO cell lines, with all tested strains (WSN, H5N1, H7N9), albeit no obvious difference in levels of viral mRNA and vRNA were found. Notably, we observed similar effect of AGO2 on virus replication in RIG-I-, Drosha- and Dicer-KO A549 infected cell lines, arguing a mechanism that the AGO2 inhibition of IAV replication is independent of IFN-b, microRNA or RNAi pathway. In addition, using cross-linking and immunopurication (CLIP) and fluorescence in situ hybridization (FISH) assays, we showed that Ago2 only associates with viral mRNA, but not with viral genomic RNA. Taken together, our data suggests an undefined role of Ago2 in suppression of IAV replication through translational regulation. Furthermore, we demonstrated that the non-structural protein (NS1) is able to bind different functional domains of Ago2 individually; and the association between effector domain (ED) of NS1 and AGO2 is RNA-independent. In the mini-genome assay, we found that the suppression of AGO2 on RNP activities takes place only in the presence of NS1 protein. The relationship between AGO2 and NS1 in viral replication will be explored in detailed.