The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3 termini ended with 5-UGAACC-3 instead of the 3 consensus sequence 5-UGACC-3, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3 consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3 consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3 end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3 consensus sequence.Rotavirus virions are icosahedral particles consisting of three layers of protein and containing 11 segments of doublestranded RNA (dsRNA) (8). The innermost protein layer has a Tϭ2 arrangement and is formed by the core lattice protein VP2 (reviewed in reference 29). Associated with the interior side of each of the 12 pentamers of the VP2 lattice is believed to be one copy each of the viral RNA-dependent RNA polymerase (RdRP) VP1 and the mRNA-capping enzyme VP3 (19,20). Based on structural studies of rotaviruses and other members of the family Reoviridae (29), it is thought that each genome segment exists as a tightly wound spiral around one of the 12 RdRP-capping complexes of the VP2 lattice (9). Collectively, the VP2 lattice, the RdRP-capping complexes, and the dsRNA genome make up the core of the virion. Doublelayered particles consisting of cores surrounded by the intermediate protein VP6 have transcriptase activity and are responsible for the synthesis of the 11 viral mRNAs (1, 37). The dsRNA genome probably exists as a liquid crystal within the core and, in this form, has the fluidity necessary for the dsRNA segments to slide through the anchored RdRP-capping enzyme complex during transcription (9). Nascent transcripts produced by the viral RdRP are extruded through channels located at the vert...

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

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