Abstract
Human noroviruses (HuNV) are the leading cause of gastroenteritis worldwide, and murine norovirus (MNV) is a useful model for studying virus-host interactions. The open reading frame 1 (ORF1) is translated during MNV infection to produce the ORF1 polyprotein that is cleaved by the viral protease (Pro) at specific protease sites to release non-structural (NS) proteins. These protease sites are not recognised equally, leading to the generation of partially processed precursor proteins. The HuNV ProPol precursor has protease substrate specificities that differ from the mature protease. However, whether the MNV ProPol has different protease targets to the Pro within the viral NS or host cell proteins has not been explored.
NS1-2 is further cleaved by caspase 3 during infection. However, non-caspase related fragments, termed NS1c-2, have also been observed. A potential viral protease site (E66/G67) was predicted in NS1-2 to produce an NS1c-2 fragment. Mass spectrometry identified G67 at the N-terminus of an NS1c-2 fragment which confirmed cleavage at the E66/G67 site. To determine what protease was responsible for cleaving E66/G67 to generate NS1c-2, proteolytically active MNV ProPol and Pro were expressed and purified. MNV ProPol cleaved NS1-2.TM at E66/G67 site, whilst MNV Pro did not.
Sequence analysis identified two variants of the NS1c-2 cleavage site within MNV strains. Three mutant viruses were generated to characterise the effect of the NS1c-2 cleavage site on MNV replication. The MNV E66A mutant was designed as the unprocessed control and showed the largest decrease in MNV replication in Huh7CD300lf and BV-2 cells, demonstrating that cleavage at this site was important to virus replication.
Actin, SUG1 and hnRNPK were identified via iTRAQ-TAILS methodology as potential cellular targets of MNV ProPol and Pro. Both proteases were shown to cleave SUG1 and hnRNPK, with ProPol cleaving hnRNPK more efficiently than Pro. Targeting of SUG1 and hnRNPK by MNV was confirmed in an infected cell. Knockdown of hnRNPK increased viral NS1-2 and RNA levels, indicating hnRNPK antagonizes MNV replication. HuNV ProPol and Pro also cleaved hnRNPK, showing a conserved mechanism among murine and human noroviruses.
This thesis provides evidence that MNV ProPol has distinct protease activity to the mature viral protease to cleave viral and cellular proteins. Mutations to the NS1c-2 cleavage site demonstrated that cleavage at this site is important for MNV replication in a human cell line and even more so in a murine cell line. This work also demonstrates the importance of MNV targeting hnRNPK by different forms of the viral protease to enhance MNV replication.