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Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

ABSTRACT: 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 sigmaNS and core protein mu2 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 sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS 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 sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.

SUBMITTER: Becker MM

PROVIDER: S-EPMC114052 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

Becker M M MM Goral M I MI Hazelton P R PR Baer G S GS Rodgers S E SE Brown E G EG Coombs K M KM Dermody T S TS

Journal of virology 20010201 3

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 sigmaNS and core protein mu2 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 sigmaNS and mu2 in viral inclusion formation. In ...[more]

PMID: 11152519

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Project description:Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein muNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of muNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue muNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of muNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of muNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in muNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with Aequorea victoria green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across muNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of muNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells.

Dataset Information

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

Publications

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

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