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ABSTRACTProgeny 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 µ2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and tsmutant reoviruses to define the roles of ?NS and µ2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, ?NS and µ2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with ?NS-mutant virustsE320, ?NS is distributed diffusely in the cytoplasm and µ2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with µ2-mutant virus tsH11.2, µ2 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 µ2 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 µ2, are recruited to commence dsRNA synthesis.

ABSTRACTPrevious studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279identified two tslesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46–58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the ?2 protein, is associated with the accumulation of a core-like particle defective for the ?2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein ?2, and the minor core protein µ2. Core particles with the ?2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins ?1, ?3, and ?2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein µ2, either preceding or collateral to the condensation of the ?2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.