Towards understanding the influenza virus ribonucleoprotein complex: novel structural and functional approaches

Thesis

Influenza viruses pose a significant public health concern, given their capacity to cause seasonal epidemics and pandemics like the infamous 1918 influenza pandemic. Central to the viral life cycle is a protein-RNA complex known as viral ribonucleoprotein (vRNP), for viral genome transcription and replication. For decades, studies focused on individual vRNP protein components, namely viral polymerase and nucleoprotein (NP), along with their regulators. Nevertheless, the ultimate goal of visualizing vRNP in various functional states at the molecular level and in real-time remains to be achieved. To progress toward this objective, establishing a minimal system that reconstitutes vRNP functions in a completely cell-free environment would be advantageous. Additionally, there is a need to image vRNPs at a resolution that allows unambiguous localisation of each vRNP component. During my DPhil, my aim was to make advancements toward this goal.

vRNPs isolated from virions transcribe vRNA in a test tube to make viral mRNA when supplied with nucleotides and a cap donor RNA. To recreate the two-step viral genome replication in a simplified and highly controlled in vitro system, I initially switched virionisolated vRNP from mRNA synthesis to cRNA synthesis by adding purified recombinant viral polymerase and host ANP32 protein. Subsequently, I reconstituted a full cycle of viral genome replication in a test tube by further including NP, promoting the second step of replication. This novel system revealed the minimal protein requirements to activate cRNA synthesis in a vRNP and to complete a full viral genome replication cycle. Through introducing mutations to the added proteins, I also managed to dissect their roles and uncover their novel functions. These findings offer original insights into the molecular regulation of vRNP functions. I then utilized a panel of nanobodies with resolved binding sites on the 1918 influenza viral polymerase to identify new inhibitory sites on the viral polymerase and unveil new antiviral mechanisms against influenza virus. Finally, using nanobodies raised against the viral polymerase or NP, I generated megabodies to help structurally resolve the interactions between viral polymerase and NP, and between NP and RNA, in the context of vRNP.

Collectively, this work provides novel insights into the molecular mechanisms of vRNP functions and introduces new approaches to study influenza virus vRNP. Specifically, it lays the groundwork for studying the regulation of vRNP functions systematically and paves the way for imaging viral replication in the context of vRNP. Excitingly, with the system and tools developed in this thesis, we are now closer than ever to visualising a functional vRNP in realtime.

Authors: Zhu, AZ

• DOI: 10.5287/ora-5jzdmmkwg
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: RNA replication; nanobody; in vitro assay; regulatory factors; influenza virus
• Subjects: Molecular virology