Reovirus mu 2 protein determines strain-specific differences in the rate of viral inclusion formation in L929 cells

Reovirus infection induces the formation of large cytoplasmic inclusions th at serve as the major site of viral assembly. Reovirus strains type 3 Deari ng (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 infect ed 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 fou nd that the Mf gene, which encodes the mu 2 protein, is the primary determi nant of the rate or inclusion formation. The S3 gene, which encodes the non structural protein sigma NS, plays a secondary role in this process. The su bcellular location of the mu 2 protein was determined by confocal laser sca nning microscopy using dual-fluorescence labeling of mu 2 and the outer-cap sid protein mu 1/mu 1C. In virus-infected cells, mu 2 protein colocalized w ith other Viral proteins in inclusions and was also distributed diffusely i n the cytoplasm and nucleus. Expression of recombinant T1L and T3D mu 2 pro teins resulted in the formation of protein complexes resembling inclusions in both the cytoplasm and the nucleus with kinetics that reflected the stra in of origin. The median time of mu 2 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 mu 2 protein influences th e rate of inclusion formation and contributes to inclusion morphogenesis. T he requirement of mu 2 protein in inclusion formation was tested by determi ning the subcellular localization of mu 2 in cells infected with temperatur e-sensitive (ts) mutants that are defective in Viral assembly. In contrast to infection with wild-type virus, mu 2 did not colocalize with mu 1/mu 1C protein in subcellular structures that formed in cells infected at nonpermi ssive temperature with ts mutants tsH11.2, tsC447, and tsG453 with mutation s in the M1, S2, and S4 genes, respectively. These results suggest that des pite the role of the mu 2 protein In controlling the rate of inclusion form ation, this process is a concerted function of several reovirus proteins. ( C) 2000 Academic Press.