Reovirus growth in cell culture does not require the full complement of viral proteins: identification of a sigma1s-null mutant.
The reovirus sigma1s 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 the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s. To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, 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 sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.
Departments of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
S E
SE
Rodgers
J L
JL
Connolly
J D
JD
Chappell
T S
TS
Dermody
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017209:Apoptosis; D001483:Base Sequence; D036022:Capsid Proteins; D002460:Cell Line; D004285:Dogs; D018507:Gene Expression Regulation, Developmental; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D014644:Genetic Variation; D007739:L Cells; D012089:Mammalian orthoreovirus 3 / Q000235:genetics* / Q000254:growth & development* / Q000502:physiology; D051379:Mice; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D012367:RNA, Viral; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus