Structural studies of viral biogenesis mechanisms

This study targets two understudied viral systems, the negative-sense ssRNA viruses (-ssRNA) and circular dsDNA membrane-containing viruses, providing insights on their functioning, in particular genome delivery, assembly and role of the structural proteins in those processes. Although much has been learned about viruses, their vast sequence diversity means that many remain understudied, with up to 60% of the global virome still constituting “viral dark matter,” comprising genes with poorly understood or unassigned functions.
Bovine ephemeral fever virus (BEFV), an -ssRNA virus from the Ephemerovirus genus within the Rhabdoviridae family, was targeted due to its economic significance and importance for animal health, and owing to its relatively distant evolutionary relationship to other members of the family, which have already been structurally characterised. The main focus of this thesis was on nucleoprotein, the key genome assembly and delivery factor. The nucleoprotein-RNA complex was produced and its structure determined using cryo-EM at 2.9 Å resolution. The structure revealed details of protein-RNA and protein-protein interactions. The insights obtained from the oligomeric assembly were used to design peptides that may inhibit the nucleoprotein oligomerisation. This project also attempted production of BEFV virus-like particles in plants for their subsequent use in structural studies and vaccine development.
The circular dsDNA membrane-containing viruses were studied using two representatives, phages Jorvik and PM2. Structure of Jorvik capsid was resolved at the resolution of 11.2 Å using cryo-ET subtomogram averaging, providing first structural insights on this phage. Structure of PM2 was determined at 2.9 Å resolution using cryo-EM single particle analysis, providing new insights on the interaction between capsid proteins, as well as virus assembly machinery. Cryo-ET studies of PM2 interaction with its host showed that virus attaches to pili filaments, whereas Jorvik attaches to the cell capsule. These studies revealed major differences in host infection, even among dsDNA viruses belonging to the same virus lineage.