Influenza A virus (IAV) is a major cause of human respiratory infections with a disease pathogenesis that varies widely depending on the strain of virus. The genome of IAV consists of eight negative sense single-stranded RNA segments. In addition to PA, which is an essential component of the viral RNA polymerase complex, segment 3 of IAV can also produce the accessory protein PA-X as a consequence of ribosomal frame-shifting during translation. PA-X consists of the 191 aa N-terminal endoribonuclease domain of PA fused to a novel 61 or 41 aa X-domain. The function of PA-X is not fully characterised. Detection of viral RNAs by the cytosolic RNA helicase RIG-I is a key trigger for type I interferon (IFN) production during IAV infection. Given that PA-X harbours an endoribonuclease domain, we hypothesised that PA-X has a role in modulating the host response to IAV infection by degrading RIG-I-stimulatory RNAs, thereby preventing activation of this pathway.

We show that overexpression of PA-X from different IAV strains diminishes IFN production in response to RIG-I-stimulatory IAV RNA in vitro, via a mechanism dependent on the endonuclease activity of PA-X. Cellular viability, protein synthesis and IFN production in response to a different RNA virus, Sendai virus, was not affected by PA-X overexpression. We further show that infection with a PA-X-deficient virus induces more IFN both in vitro and in vivo, and that this is dependent on the presence of the RIG-I signalling adaptor protein MAVS. We therefore conclude that the evolutionarily conserved viral protein PA-X specifically acts to limit activation of the RIG-I pathway by IAV RNA. This observation may have important implications for IAV disease pathogenesis and transmission, given that cytokine responses can be both beneficial to the host by promoting clearance of the virus as well as contributing to immunopathology.