Reovirus has a segmented dsRNA genome enclosed in a nonenveloped double capsid shell. Reovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site for viral assembly. These inclusions contain the intermediate filament vimentin, in addition to viral proteins, RNA, mature and immature viral particles. However, the viral or host proteins involved in the formation of reovirus inclusions have not been identified, neither has the mechanism by which viral RNA and proteins are directed to these sites. In this study the reovirus M1 gene, which encodes the minor core protein mu2, was identified as the primary determinant of the rate of inclusion formation. Cellular localization studies of mu2 protein in cells infected with wild-type or ts mutants of reovirus as well as in M1 gene-transfected cells showed that mu2 protein may directly be involved in inclusion formation. Expression of mu2 protein in infected cells was shown to be strain-dependent with type 1 strain "Lang" (T1L) producing significantly more mu2 protein than type 3 strain "Dearing" (T3D). Protein stability was demonstrated to be partly responsible for the difference in mu2 protein expression between T1L and T3D, with T1Lmu2 being more stable than T3D mu2. Degradation of mu2 protein was shown to occur via the ubiquitin-proteasome pathway (UPP). Ubiquitinated mu2 protein was present in reovirus inclusions together with components of the UPP making reovirus inclusions structurally similar to aggresomes and suggesting that reovirus may be using the UPP and aggresome formation pathway for inclusion formation. In keeping with this theory, it is shown that both reovirus inclusion formation and replication are inhibited when the cellular pool of free ubiquitin (Ub) is depleted by using proteasome inhibitor and that replication maybe enhanced by mono-ubiquitination. Interestingly, the most abundant ubiquitinated mu2 protein species was mono-ubiquitinated mu2 suggesting a role for mono-ubiquitinated mu2 in reovirus inclusion formation and viral assembly. In addition, the amino-terminal end of recombinant mu2 protein, which contains a potential E3 Ub-ligase motif, was shown to be important for protein complex formation. Most importantly reovirus inclusions, like aggresomes, were shown to impair the normal functioning of the UPP resulting in inhibition of Ub-dependent proteolysis which presumably contributes to cytopathology and disease in infected animals. This study is the first to show the use of the UPP in the replication of a nonenveloped or dsRNA virus.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/6267 |
| Date | January 2002 |
| Creators | Mbisa, Jean Lutamyo. |
| Contributors | Brown, Earl G. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 183 p. |
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