REOVIRUS GROWTH IN CELL-CULTURE DOES NOT REQUIRE THE FULL COMPLEMENT OF VIRAL-PROTEINS - IDENTIFICATION OF A SIGMA-1S-NULL MUTANT

The reovirus als protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis, Although th e function of als is not known, the als open reading frame is conserve d in all S1 gene sequences determined to date. In this study, we ident ified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express als, To facilitate these experiments, we generated tw o monoclonal antibodies (MAbs) that bind different epitopes of the als protein. Using these MAbs in immunoblot and immunofluorescence assays , we found that L929 (L) cells infected with T3C84-MA do not contain a ls, To determine whether als is required for reovirus infection of cul tured cells, we compared the growth of T3C84-MA and its parental strai n, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells . After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether als is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were t ested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic ce lls following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein als is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does no t require the full complement of virally encoded proteins.