Previous reports revealed that the M3 gene of both avian and mammalian reoviruses express two isoforms of the non-structural protein mNS in infected cells. The larger isoforms initiate translation at the AUG codon closest to the 59 end of their respective m3 mRNAs, and were therefore designated mNS. In this study we have performed experiments to identify the molecular mechanisms by which the smaller mNS isoforms are generated. The results of this study confirmed the previous findings indicating that the smaller mammalian reovirus mNS isoform is a primary translation product, the translation of which is initiated at the internal AUG-41 codon of mammalian reovirus m3 mRNA. Our results further revealed that the smaller avian reovirus mNS isoform originates from a specific post-translational cleavage site near the amino terminus of mNS. This cleavage produces a 55 kDa carboxy-terminal protein, termed mNSC, and a 17 kDa amino-terminal polypeptide, designated mNSN. These results allowed us to extend the known avian reovirus protein-encoding capacity to 18 proteins, 12 of which are structural proteins and six of which are non-structural proteins. Our finding that avian and mammalian reoviruses use different mechanisms to express their mNSC isoforms suggests that these isoforms are important for reovirus replication.
Avian reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of avian reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particles.
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