Insights into viral RNA synthesis by the influenza A virus and SARS-CoV-2 RNA polymerases

Influenza A virus and SARS-CoV-2 are RNA viruses which cause severe respiratory disease and death in humans. Both encode RNA-dependent RNA polymerases (RdRps), which are responsible for synthesising viral RNA as part of a larger complex of viral proteins; for influenza A virus this complex is called a ribonucleoprotein (RNP), and for SARS-CoV-2 the replication- transcription complex (RTC). In this work I elucidate mechanisms used by influenza A virus and SARS-CoV-2 to synthesise viral RNA. I first present evidence that the influenza A virus RdRp maintains the structural integrity of RNPs during transcription by binding to the 3′ end of viral genomic RNA. I then go on to demonstrate that in order to initiate viral RNA synthesis, the influenza A virus RdRp requires trans-activation through dimerization. Furthermore, I show that the influenza A virus RdRp co-opts the host protein ANP32A to help it form a second structurally-distinct dimer, which has the role of encapsidating nascent viral RNA into progeny RNPs. Collectively, these findings allow me to present a more detailed molecular model for how influenza viral RNA is synthesised and assembled into new RNPs. One role of the influenza A virus RNP is to hide highly immunogenic viral RNA from cellular innate immune receptors such as RIG-I. The SARS-CoV-2 RTC instead synthesises 7- methylguanosine (m7G)-capped viral RNA which does not activate RIG-I. In this work I also show that one of the domains in the SARS-CoV-2 RdRp is a guanylyltransferase enzyme, which resolves a missing link in the mechanism of coronavirus m7G cap synthesis. Together, these studies improve understanding of the diverse mechanisms used by RNA viruses to synthesise viral RNA, and identify multiple functionally important sites on the influenza A virus and SARS- CoV-2 RdRps which could be exploited for future antiviral strategies.