Influenza A viruses (IAV) are negative-strand segmented RNA viruses and are responsible for causing severe respiratory diseases and fatalities in humans. IAV encodes an RNA-dependent RNA polymerase (FluPolA), which is responsible for transcribing and replicating each segment of the viral genome. In addition to the full- length vRNAs and cRNAs, the FluPolA synthesizes aberrant RNA products, such as defective interfering RNAs (diRNAs) and mini viral RNAs (mvRNAs).

The innate immune system serves as the body's primary defence against viruses. Within this intricate defence network, RIG-I is responsible for detecting viral RNAs in the host cell, initiating a signalling cascade that ultimately triggers the expression of interferons and innate immunity. Adding to this complexity, IFI16, another essential cellular factor, is recognized for its ability to enhance the binding of specific full-length viral segments to RIG-I and increase RIG-I signalling.

In this study, I set out to understand what factors determine which IAV RNAs are RIG-I agonists. Additionally, I sought to determine whether IFI16 demonstrates analogous behaviour when engaging with mvRNAs as it does with full-length viral segments. Finally, I aimed to improve our knowledge regarding the factors that govern transcription and replication processes.

Here, I present an RNA structure-dependent (t-loop) innate immune activation mechanism that potentially underlies how mvRNAs induce RIG-I-dependent signalling. T-loop-containing mvRNAs are poorly replicated and strong t-loops are even capable of FluPolA stalling. Further experiments show that FluPolA stalling does not result in release of the mvRNA templates or nascent RNAs, but instead I observe that the stalled mvRNA- FluPolA complex itself activates innate immune signalling in a manner which seems to be dependent on the helicase activity of RIG-I. I also observe that, in contrast to mvRNAs, the full-length IAV segments can form interactions with IFI16 in a length-dependent manner. It also appears that IFI16 competes with RIG-I for binding to the NA RNA segment.

Additionally, mvRNA transcription can lead to the production of an additional aberrant product; a cRNA with a cap1 structure (ccRNA), which appears to interact with RIG-I. This aberrant RNA is the result of a defect during polyadenylation. Finally, this study provides evidence that both the PB1 C-terminal residues and priming loop residues play a critical role at every stage of IAV transcription initiation. Moreover, these PB1 C-terminal residues also exert a significant influence over the replication process.

Overall, my research contributes to a better understanding of the mechanisms that underlie the innate immune response to IAV and sheds light on the roles of different viral components in the transcription and replication of the viral genome.