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.
To better understand genetic diversity of mammalian reoviruses, we studied sequence variability in the S3 gene segment of 17 field-isolate reovirus strains and prototype strains of the three reovirus serotypes. Strains studied were isolated over a 37-year period from different mammalian hosts and geographic locations. A high degree of variability was observed in the nucleotide sequences of the S3 gene, whereas the deduced amino acid sequences of the S3 gene product, sigma NS, were highly conserved. When variability among the S3 nucleotide sequences was analyzed using pairwise comparisons, we found that 5' and 3' noncoding regions were significantly more conserved than the remainder of the gene. This high degree of sequence conservation was also observed within the first 15 nucleotides of the 5' coding region. Phylogenetic analyses showed that multiple alleles of the S3 gene cocirculate and that genetic diversity in the S3 gene does not correlate with host species, geographic locale, or date of isolation. Phylogenetic trees constructed from variation in the S3 sequences are distinct from those previously generated from sequences that encode attachment protein sigma 1, core protein sigma 2, and outer capsid protein sigma 3, which supports the hypothesis that reovirus gene segments reassort in nature. These findings suggest that reovirus gene segments are well-adapted to mammalian hosts and that reovirus evolution has reached an equilibrium.
To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced r2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and af2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from Si and S2 gene nucleotide seqiwnces of the seven type 3 strains. These results demonstrate that reovirus Si and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 ff2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other cr2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced ff2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of fr2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.
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