Caliciviruses are single-stranded positive-sense RNA viruses, most commonly
associated with outbreaks of gastroenteritis in humans. In addition to the three open
reading frames (ORFs) typical of caliciviruses a highly conserved fourth overlapping
ORF within the murine norovirus (MNV) genome was identified and characterised
during this project. ORF4 overlaps and is contained within the capsid encoding ORF2
in a +1 frame. Once a suitable antibody had been generated, immunoblotting was used
to show that the ORF4 protein is produced during MNV infection. Although ORF4
mutant viruses were viable and replicated to wild-type MNV levels in tissue culture,
pressure to restore ORF4 expression upon serial passage under specific conditions was
demonstrated. Importantly, the ORF4 knockout virus was significantly attenuated in
STAT1-/- mice. Proteomic analysis and a commercial yeast two-hybrid screen were
used to identify host cell factors which interact with the ORF4 protein and confocal
imaging was employed to examine cellular localisation. These approaches indicated
that the ORF4 protein localises to the mitochondria and in vitro assays were
subsequently used to demonstrate an involvement of the ORF4 protein in MNV
induced apoptosis. As cells infected with the knockout virus showed an earlier and
higher degree of apoptosis induction compared to wild-type infected cells, it is
possible that the ORF4 protein may function to delay the onset of apoptosis during
MNV infection. Whether or not the ORF4 protein has antiapoptotic activity or whether
the difference in apoptotic phenotype is an indirect consequence of ORF4 protein
function remains to be investigated. These data indicate that the ORF4 protein
represents a novel, previously uncharacterised virulence determinant in MNV
