Investigating the mechanisms behind the immunostimulatory activity of influenza A virus RNA polymerase mutations

Activation of innate immune signalling during influenza A virus (IAV) infection is essential for the development of protective antiviral responses. However, uncontrolled release of proinflammatory cytokines is also associated with severe and fatal disease caused by highly pathogenic avian and pandemic IAV strains. IAV infections are primarily detected by the retinoic acid-inducible gene I (RIG-I), which recognises the partially double-stranded, 5'-triphosphorylated RNA structure that is formed by the protein-free 3' and 5' termini (viral promoter) of viral genomic segments as well as aberrant replication products. However, it remains unknown how RIG-I gains access to the viral promoter when it is bound by the viral polymerase, and if other viral RNA species could play a role in RIG-I activation. The viral RNA polymerase itself was shown to act as both an innate immune agonist by producing immunostimulatory RNA species, and as an antagonist by directly interacting with the components of the RIG-I signalling pathway. However, the mechanisms underlying these roles of the viral polymerase in the IAV infection cycle are not completely understood. In this thesis, I investigated the mechanisms behind the immunostimulatory activity of two mutations (D27N and T677A) in the polymerase basic 1 (PB1) subunit that were identified in a single-cell sequencing screen. Viruses containing the PB1 D27N and T677A mutations activated interferon (IFN) responses via the RIG-I signalling pathway. The immunostimulatory activity of the T677A mutation was also recapitulated in an *in vitro* ribonucleoprotein reconstitution assay. Surprisingly, IFN induction by the T677A polymerase was to a large extent driven by its transcriptional activity. Co-immunoprecipitation assays showed that RIG-I bound capped positive-sense RNA produced by the T677A polymerase. This finding suggests the existence of a novel mechanism by which defects in viral RNA synthesis activate innate immune signalling during viral infection. The D27N and T677A viruses were also severely attenuated in cell culture. Reduced fitness of the D27N virus was partly associated with the defects in segment 6 packaging, which led to low levels of neuraminidase expression and impaired haemagglutinin maturation. Adaptation of the T677A and D27N viruses during serial passaging suggested that changes in other viral proteins compensated for the fitness defects created by the PB1 mutations. These changes could be further explored to understand the interplay between the RNA polymerase and other viral proteins at different stages of the viral life cycle.