Post-transcriptional regulation of Influenza A virus mRNAs

Abstract

The fate of cellular mRNAs is governed by intricate and dynamic post-transcriptional regulatory mechanisms that fine-tune cellular gene expression. Processes such as stabilisation, degradation, splicing, nuclear export and RNA editing, are essential for mRNA processing and are known to be hijacked by RNA viruses to enhance their own gene expression. It is well established that influenza A virus (IAV) exploits these pathways as a means to increase viral gene expression, replication and pathogenesis. However, many questions remain surrounding the precise mechanisms behind these interactions. The work presented in this thesis reveals novel insights into mechanisms driving IAV mRNA post-transcriptional regulation. Chapter 1 begins with a review of post-transcriptional regulatory processes utilised by four enveloped respiratory viruses: Influenza A virus (IAV), Human Parainfluenza virus (hPIV), Respiratory Syncytial virus (RSV) and Severe Respiratory Coronavirus-2 (SARS-CoV-2). Chapter 2 presents an investigation into the epitranscriptomic landscape of IAV mRNAs, with a focus on N-6-methyladenosine (m6A) and pseudouridine (Ψ), two of the more abundant RNA modifications for cellular mRNA. Internal base modifications are dynamic regulators of RNA and play crucial roles during the lifecycle of both cellular and viral RNA. We used single-base resolution mapping techniques DART-Seq and BID-seq, to map m6A and Ψ, respectively, on IAV mRNAs. We uncovered several high-confidence m6A sites across IAV mRNAs, while conversely finding no Ψ sites across IAV mRNAs, providing interesting insights into epitranscriptomic regulation of IAV mRNAs. However, RNA modifications represent a small fraction of post-transcriptional regulation. Manipulation of host cell RNA binding proteins (RBPs) is vital for productive flu infection, as these interactions are important for several post-transcriptional regulatory processes. As IAV is a nuclear replicating RNA virus one critical aspect of post-transcriptional regulation is the export of viral mRNAs from the nucleus to the cytoplasm. Chapter 3 focuses on a novel IAV mRNA nuclear export factor, MKRN2, and its proviral role in the IAV lifecycle. This data sheds light on an important aspect of IAV infection and offers new opportunities for the exploration of MKRN2’s interacting partners during viral infection. In Chapter 4 I go onto identify and describe the role of the cold-inducible protein, RBM3, as a stabilising agent of viral nucleoprotein (NP) mRNA. Our findings around this pro-viral interaction have important implications for other respiratory viruses, such as SARS-CoV-2 and RSV, and the potential developments of future mRNA-based aerosolized vaccines, which may be designed to exploit the stabilising function of RBM3. Overall, this body of work presents a diverse investigation into several post-transcriptional regulatory mechanisms of IAV mRNAs and provides crucial insights into the RNA biology of IAV.