The coronavirus genome is a positive-stranded RNA of extraordinary size and complexity. The largest protein encoded by coronaviruses is non-structural protein 3 (nsp3), which contains many domains of unknown function. In this study, a vaccinia virus-based reverse genetics system was used to introduce seven mutations in the C-terminal domain of nsp3, known as the Y domain. Of these, four recombinant MHVs were constructed successfully. However, none of them was rescued, suggesting that the Y domain performs an important function in the MHV replication cycle. The function of nsp3 in replication was then probed by biochemical and structural methods using the temperature-sensitive mutant Brts31 which contains a mutation in nsp3 along with four other temperature-sensitive mutants that contained mutations in each of the other cistrons that function in viral RNA synthesis. Interestingly, Brts31 and several other viruses tested formed smaller and fewer DMVs compared to the parental wild-type virus under conditions where both viruses produced an equal amount of progeny virus. This suggested that the efficiency of producing progeny virus is not closely related to the prevalence or size of DMVs. Two MHV mutants (Brts31 and Brts105) made half as many DMVs as normal. Despite differences in DMV size and number, all mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelle formation , we carried out competitive fitness experiments using viruses with different DMV phenotypes. None of these viruses was found to be significantly less fit than wildtype, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane associated replication complexes, and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:629089 |
| Date | January 2014 |
| Creators | Al-Mulla, Hawaa M. N. |
| Publisher | University of Reading |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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