Transcriptional activities of reovirus RNA polymerase in recoated cores. Initiation and elongation are regulated by separate mechanisms.

The particle-associated reovirus polymerase synthesizes mRNA within only certain viral particle types. Reovirus cores, subviral particles lacking outer capsid proteins mu1, sigma3, and sigma1, produce mRNA and abortive transcripts. Reovirus virions, which contain complete outer capsids, cannot produce mRNA and produce few abortive transcripts. Recoated cores are virion-like particles generated by the addition of recombinant outer capsid proteins to cores. We used recoated cores to analyze transcriptional regulation by reovirus outer capsid proteins. Partially recoated particles, containing less than virion amounts of mu1 and sigma3, synthesized mRNA at levels inversely proportional to outer capsid protein levels. Fully recoated cores exhibited undetectable mRNA synthesis levels, as did virions. However, recoated cores produced high levels of abortive transcripts. Recoated core abortive transcripts remained particle-associated and appeared to inhibit further abortive transcript production. Proteolysis of recoated cores removing mu1 and sigma3 released accumulated abortive transcripts and relieved inhibition of mRNA and abortive transcript synthesis. These results suggest transcriptional elongation, but not initiation, is blocked by virion-like amounts of mu1 and sigma3. Particle-associated abortive transcripts may down-regulate transcriptional initiation. Minor outer capsid protein sigma1 had no demonstrable effect on transcriptional activities. Transcriptional regulation may ensure progeny virions do not compete with transcribing particles for ribonucleoside triphosphates.

Mammalian reovirus L3 gene sequences and evidence for a distinct amino-terminal region of the lambda1 protein.

To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5' triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein lambda1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the lambda1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced lambda1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The lambda1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the lambda1 open reading frame to within 60 nucleotides of the plus-strand 3' end for T3D and the other isolates sequenced, in keeping with the short 3' nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D lambda1 were located within the amino-terminal 500 residues of lambda1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and lambda1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of lambda1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in lambda1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure-function relationships for the lambda1 core protein.

Binding site for S-adenosyl-L-methionine in a central region of mammalian reovirus lambda2 protein. Evidence for activities in mRNA cap methylation.

One or more proteins in mammalian reovirus core particles mediate two RNA methylation activities, (guanosine-7-N)-methyltransferase and (guanosine-2'-O)-methyltransferase, that contribute to forming the 5' cap 1 structure on viral mRNA. We used UV irradiation to identify core proteins that bind S-adenosyl-L-methionine (SAM), the methyl-group donor for both methyltransferases. A [methyl-3H]SAM-binding site was observed among the reovirus lambda proteins; was shown to be specific by competition with low levels of S-adenosyl-L-homocysteine, the product of methyl-group transfer from SAM; and was subsequently localized to protein lambda2. lambda2 mediates the guanylyltransferase reaction in cap formation and was previously proposed to mediate one or both methylation reactions as well. SAM binding was demonstrated for both lambda2 in cores and lambda2 expressed in insect cells from a recombinant baculovirus. Using three different methods to cleave lambda2, a binding site for SAM was tentatively localized to a central region of lambda2, between residues 792 and 1100, which includes a smaller region with sequence similarity to the SAM-binding pocket of other methyltransferases. Alanine substitutions at positions 827 and 829 within this predicted binding region greatly reduced the capacity of baculovirus-expressed lambda2 protein to undergo UV cross-linking to SAM but had no effects on either the guanylyltransferase activity of this protein or its conformation as judged by partial proteolysis, suggesting that one or both of these residues is essential for SAM binding. Based on these findings, we propose that the two methyltransferase activities involved in mRNA capping by reovirus cores utilize a single SAM-binding pocket within a central region of lambda2.

Internal/structures containing transcriptase-related proteins in top component particles of mammalian orthoreovirus.

The structure of mammalian orthoreovirus top component particles, which are profoundly deficient in the content of double-stranded RNA genome, was determined at 30 A resolution by transmission cryoelectron microscopy and three-dimensional image reconstruction. Previously undetected, ordered densities, appearing primarily as pentameric flowers in the reconstruction, were seen to extend 65 A inwardly from the inner capsid at the icosahedral fivefold axes. Identically positioned but lower density elements were observed in two types of partially uncoated top component particles obtained by limited proteolysis. The levels of three inner-capsid proteins-lamda 1, lamda 3, and mu 2-were reduced in concert with the internal densities during proteolytic uncoating. Since lamda 3 contains the catalytic regions of the viral RNA polymerase and since both lamda 1 and mu 2 appear to play roles in transcription or mRNA capping, the internal structures are concluded to be complexes of the viral transcriptase-related enzymes. The findings have implications for the mechanisms of transcription and mRNA capping by orthoreovirus particles.

Localization of a C-terminal region of lambda2 protein in reovirus cores.

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.