AbstractRabbit caliciviruses belong to the family Caliciviridae, genus Lagovirus and are used in Australia to manage the numbers of European rabbits (Oryctolagus cuniculus), introduced vertebrate pest species. Feral rabbit populations damage the environment leading to extinction of native plant and animal species and causing agricultural losses. The Rabbit haemorrhagic disease virus (RHDV) is a pathogenic lagovirus that causes liver failure and haemorrhages in adult, but not young rabbits. Another lagovirus, RHDV2, frequently kills both adult and young rabbits. These viruses are thought to have evolved from non-pathogenic rabbit caliciviruses that infect intestinal, and not liver tissues. In the Iberian Peninsula, lagomorphs are important elements of the environment and farmed rabbits represent a food source. RHDV outbreaks in farmed rabbits are not desirable and rabbit vaccination is implemented. Despite the use of lagoviruses as a biocontrol agent in Australia and efforts to prevent outbreaks in Europe, many aspects of their virology remain poorly understood. This is mostly due to the inability of researchers to grow rabbit caliciviruses in the laboratory, as they cannot be propagated in a conventional cell culture system. Recent advances towards organoid culture systems may soon improve our knowledge in this area.
Caliciviridae have a small positive-sense single-stranded RNA genome that encodes structural (capsid) and non-structural proteins. The non-structural proteins are responsible for virus replication and modification of the host cell to accommodate virus replication. Thus, these proteins are likely important virulence determinants of lagoviruses. For example, one of the previously discovered virulence factors is the RNA-dependent RNA polymerase (RdRp). This enzyme replicates the genome and was shown to rearrange Golgi membranes in transfected cells. The crystal structure of RdRp for most caliciviruses had been resolved and all the functional RNA polymerase motifs have been identified. In this work, however, we described a novel motif that is characteristic for caliciviruses and picornaviruses. This motif (termed motif I) was found to be conserved across the order Picornavirales. The exact function of this motif is yet to be identified.
Another knowledge gap in lagovirus virology is the unknown function(s) of several non-structural proteins of lagoviruses, namely p16, p23 and p29. I used various in vitro and in silico approaches to study these proteins, e.g., their localisation in transfected cells and presence of functional motifs in their sequences. Currently, the most studied protein of the three is p23. This protein was shown to localise to the cytoplasm and oligomerise. Adding to this information, I showed that this protein has two amphipathic helices at the C-terminus, that may form a channel upon the oligomerisation of p23. Moreover, this protein interacts with heat shock proteins (Hsp70 and 110), which may assist its correct folding and oligomerisation process. The exact localisation of this protein was also demonstrated in this study, suggesting that p23 forms channels in the endoplasmic reticulum (ER). Remarkably, cytoplasmic Ca2+ concentration was increased in p23 transfected cells, further supporting my hypothesis that p23 is a viroporin, as the ER is known to be the most important cellular Ca2+ depot.
|Date of Award||2022|
|Supervisor||Michael Frese (Supervisor), Reena Ghildyal (Supervisor), Tanja Strive (Supervisor) & Robyn Hall (Supervisor)|