Mechanisms underlying nucleolar localization of H7N9 Influenza A virus protein PB1-F2 and its role in antiviral signalling

Abstract

The emergence of H7N9 avian Influenza A virus (IAV) in 2013 is reported to cause zoonotic infection in humans. Human infection of H7N9 avian Influenza A virus has caused high morbidity rate and is clinically associated with pneumonia, multi organ failure and dysregulation of innate immunity. Understanding the underlying mechanism of innate immune suppression of H7N9 Influenza A virus would be critical for disease management. Previous work has established H7N9 PB1-F2 as a potent antagonist in type I IFN response and proinflammatory response. Interestingly, H7N9 PB1-F2 also possessed a remarkable ability to form insoluble protein aggregates. We hypothesized that H7N9 PB1-F2 protein aggregates impairs cellular quality control machinery to antagonize host innate immune response, we aimed to identify the functional role of H7N9 PB1-F2 in infection and provide the mechanistic basis of the pathogenesis of H7N9 PB1-F2 in infection to suggest new molecular targets as antivirals against H7N9 IAV infection. In the current study, H7N9 PB1-F2 was identified to localize to nucleolus by immunofluorescence staining. Live cell imaging revealed that localization of H7N9 PB1-F2 to the nucleolus markedly reduced the mobility of nucleolus implicating that nucleolar PB1-F2 as insoluble protein aggregates. Inhibition of proteasome or HSP70 activity promoted the nucleolar targeting of H7N9 PB1-F2 lending further evidence that nucleolar localization of H7N9 PB1-F2 was regulated by HSP70-mediated protein quality control pathway. In proximity labelling screen, HSP70 member HSPA14 was identified as a top hit as a proximal partner of H7N9 PB1-F2. Depletion of HSPA14 by siRNA reduced the nucleolar localization of H7N9 PB1-F2 revealing the role of HSPA14 in transporting cytoplasmic H7N9 PB1-F2 into nucleolus. Functionally, H7N9 PB1-F2 inhibited type I IFN signalling in infection. Interestingly, H7N9 PB1-F2 did not inhibit RIG-I-like receptor signalling pathway but inhibited cGAS-STING-mediated type I IFN response. Loss of HSPA14 partially rescued its inhibition on DNA sensing. Taken together, my work elucidated the molecular mechanism of nucleolar targeting of H7N9 PB1-F2 and its role as an antagonist of cGAS-STING pathway. Future work on the relevance of cytosolic DNA sensing in IAV infection, and the molecular basis of nucleolar protein quality control and cytosolic DNA release would derive important knowledge in innate immunity and antiviral strategies in highly pathogenic IAV infection.