Getting to grips with AAA+ ATPases encoded by positive-strand RNA viruses

Noroviruses (NoVs) and enteroviruses (EVs) cause numerous gastrointestinal and respiratory infections. Their contagious nature and high mutation rates can lead to the emergence of new pathogenic strains. Understanding these viruses is crucial for controlling outbreaks and to maintain global health. Both NoVs and EVs are members of the order Picornavirales and share commonalities in their replication strategies.

Their positive-strand RNA genomes encode non-structural proteins (NSPs) that exploit host cells to create membranous replication organelles for genome replication and virion formation. The most enigmatic NSP encoded by these viruses is a AAA+ ATPase (NoV NS3 and EV 2C).

These enzymes have many proposed roles in the virus lifecycle, such as RNA helicase/chaperone activity, membrane rearrangement, and genome encapsidation.

However, their structure and the molecular basis for their diverse activities are poorly understood due to challenges in producing stable and soluble complexes for structural analysis and the lack of tools to accurately visualize NSPs in a cellular context.

Drawing from my decade-long experience in structural virology and recent success in engineering soluble AAA+ ATPases, I aim to unravel the multiple functions of NoV NS3 and EV 2C.

Specifically, I will: 1) Decipher the structure-function relationship of these enzymes through single-particle cryo-electron microscopy analysis of the nucleotide and RNA-bound complexes. 2) Create a toolkit of small molecules and nanobodies to investigate the functional sites in NS3. 3) Utilize cryo-electron tomography to visualise NS3 and 2C in infected cells, guided by genetically encoded tags developed in this project.

Collectively, the results of this project will provide new mechanistic insights into the functions that NoV and EV AAA+ ATPases carry out during the viral lifecycle and pave the way for the development of novel antiviral compounds capable of inhibiting these functionally indispensable enzymes.