Reovirus μ2 Protein Determines Strain-Specific Differences in the Rate of Viral Inclusion Formation in L929 Cells

Abstract

Reovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site of viral assembly. Reovirus strains type 3 Dearing (T3D) and type 1 Lang (T1L) differ in the rate of inclusion formation in L929 cells. The median time of inclusion formation is 18 h in cells infected with T3D and 39 h in cells infected with T1L. Using reassortant viruses that contain combinations of gene segments derived from T1L and T3D, we found that the M1 gene, which encodes the μ2 protein, is the primary determinant of the rate of inclusion formation. The S3 gene, which encodes the nonstructural protein ςNS, plays a secondary role in this process. The subcellular location of the μ2 protein was determined by confocal laser scanning microscopy using dual-fluorescence labeling of μ2 and the outer-capsid protein μ1/μ1C. In virus-infected cells, μ2 protein colocalized with other viral proteins in inclusions and was also distributed diffusely in the cytoplasm and nucleus. Expression of recombinant T1L and T3D μ2 proteins resulted in the formation of protein complexes resembling inclusions in both the cytoplasm and the nucleus with kinetics that reflected the strain of origin. The median time of μ2 protein complex formation was 22 h in cells transfected with the T3D M1 gene and 43 h in cells transfected with the T1L M1 gene. These findings suggest that the μ2 protein influences the rate of inclusion formation and contributes to inclusion morphogenesis. The requirement of μ2 protein in inclusion formation was tested by determining the subcellular localization of μ2 in cells infected with temperature-sensitive (ts) mutants that are defective in viral assembly. In contrast to infection with wild-type virus, μ2 did not colocalize with μ1/μ1C protein in subcellular structures that formed in cells infected at nonpermissive temperature with ts mutants tsH11.2, tsC447, and tsG453 with mutations in the M1, S2, and S4 genes, respectively. These results suggest that despite the role of the μ2 protein in controlling the rate of inclusion formation, this process is a concerted function of several reovirus proteins.