Nonstructural Protein σ1s Mediates Reovirus-Induced Cell Cycle Arrest and Apoptosis

Reovirus nonstructural protein sigma 1s is implicated in cell cycle arrest at the G(2)/M boundary and induction of apoptosis. However, the contribution of sigma 1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of sigma 1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a sigma 1s-null reovirus. Following infection with wildtype virus, we observed an increase in the percentage of cells in G(2)/M, whereas the proportion of cells in G(2)/M following infection with the sigma 1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the sigma 1s-null mutant. These data indicate that sigma 1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in sigma 1s that mediate these effects, we engineered viruses encoding C-terminal sigma 1s truncations by introducing stop codons in the sigma 1s open reading frame. We also generated viruses in which charged residues near the sigma 1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in sigma 1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the sigma 1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.