Previous studies provided evidence that nonstructural protein NS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68: [455][456][457][458][459][460][461][462][463][464][465][466] 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of NS. Given the possible role of NS in particle assembly and/or transcription implied by those findings, we tested whether recombinant NS can bind to cores in vitro. The NS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of NS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of NS were bound. Thus, the NS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the NS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to NS. The NS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5 ends of the transcripts. In vitro competition assays showed that mixing NS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins 1 and 3. These findings are consistent with the presence of NS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, NS may block or delay outer-capsid assembly and allow continued transcription by these particles.

Nucleotide sequences of the mammalian orthoreovirus (reovirus) type 1 Lang and type 2 Jones M3 gene segments were newly determined. The nucleotide sequence of the reovirus type 3 Dearing M3 segment also was determined to compare with a previously reported M3 sequence for that isolate. Comparisons showed Lang and Dearing M3 to be more closely related than either was to Jones M3, consistent with previous findings for other reovirus gene segments. The microNS protein sequences deduced from each M3 segment were shown to be related in a similar pattern as the respective nucleotide sequences and to contain several regions of greater or less than average variability among the three isolates. Identification of conserved methionine codons near the 5' ends of the Lang, Jones, and Dearing M3 plus strands lent support to the hypothesis that microNSC, a smaller protein also encoded by M3, arises by translation initiation from a downstream methionine codon within the same open reading frame as microNS. Other analyses of the deduced protein sequences indicated that regions within the carboxyl-terminal third of microNS and microNSC from each isolate have a propensity to form alpha-helical coiled coils, most likely coiled-coil dimers. The new sequences will augment further studies on microNS and microNSC structure and function.

The reovirus L2 genome segment encodes the core spike protein lambda2, which mediates enzymatic reactions in 5' capping of the viral plus-strand transcripts. Complete nucleotide-sequence determinations were made for the L2 genome segments of eight mammalian reoviruses, including the prototype isolates of serotypes 1 and 2: Lang (T1L) and Jones (T2J), respectively. Each L2 segment was found to be 3912 or 3915 bases in length. Partial nucleotide-sequence determinations were also made for the 3916-base L2 segment of reovirus type 3 Dearing (T3D), the prototype isolate of serotype 3. The whole-genome sequence of reovirus T3D was reported previously. The T1L L2 analysis represents completion of the whole-genome sequence of that isolate as well. The T2J L2 analysis leaves only the sequence of the M1 segment yet to be reported from the genome of that isolate. The T2J M1 sequence made available from analysis in another lab was used for initiating whole-genome comparisons of reoviruses T1L, T2J, and T3D in this report. The nine L2 gene sequences and deduced lambda2 protein sequences were used to gain further insights into the biological variability, structure, and functions of lambda2 through comparisons of the sequences and reference to the crystal structure of core-bound lambda2. Phylogenetic comparisons suggest the presence of three evolutionary lines of divergent L2 alleles among the nine isolates. Localized regions of conserved amino acids in the lambda2 crystal structure include active-site clefts of the RNA capping enzyme domains, sites of interactions between lambda2 domains within the pentameric spike structure, and sites of interaction between lambda2 subunits and other proteins in viral particles.