Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L ؋ T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein 1 and the non-virionassociated protein 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that 1, rather than 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing.
Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein NS and core protein 2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of NS and 2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, NS and 2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with NS-mutant virus tsE320, NS is distributed diffusely in the cytoplasm and 2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with 2-mutant virus tsH11.2, 2 is distributed diffusely in the cytoplasm and the nucleus. However, NS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that NS precedes 2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing NS nucleate sites of viral replication to which other viral proteins, including 2, are recruited to commence dsRNA synthesis.
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