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Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

ABSTRACT: 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.

SUBMITTER: Patton JT

PROVIDER: S-EPMC230270 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Patton J T JT Jones M T MT Kalbach A N AN He Y W YW Xiaobo J J

Journal of virology 19971201 12

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, recomb ...[more]

PMID: 9371626

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Project description:The rotavirus polymerase VP1 mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein VP2. Transcription (positive-strand RNA [+RNA] synthesis) by VP1 occurs within double-layered particles (DLPs), while genome replication (double-stranded RNA [dsRNA] synthesis) by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral 5-fold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264 to 267, 547 to 550, 614 to 620, 968 to 980, and 1022 to 1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 with regard to polymerase activity. We engineered 19 recombinant VP1 (rVP1) proteins that contained single- or multipoint alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their nonmutated contact sites. These results move us toward a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation.IMPORTANCE Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase VP1 mediates all stages of viral RNA synthesis, and it requires the core shell protein VP2 for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance the understanding of a key step of the rotavirus replication cycle.

Project description:The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.

Project description:Group A rotaviruses (RVAs) are classified according to a nucleotide sequence-based system that assigns a genotype to each of the 11 double-stranded RNA (dsRNA) genome segments. For the segment encoding the VP1 polymerase, 22 genotypes (R1 to R22) are defined with an 83% nucleotide identity cutoff value. For the segment encoding the VP2 core shell protein, which is a functional VP1-binding partner, 20 genotypes (C1 to C20) are defined with an 84% nucleotide identity cutoff value. However, the extent to which the VP1 and VP2 proteins encoded by these genotypes differ in their sequences or interactions has not been described. Here, we sought to (i) delineate the relationships and sites of variation for VP1 and VP2 proteins belonging to the known RVA genotypes and (ii) correlate intergenotypic sequence diversity with functional VP1-VP2 interaction(s) during dsRNA synthesis. Using bioinformatic approaches, we revealed which VP1 and VP2 genotypes encode divergent proteins and identified the positional locations of amino acid changes in the context of known structural domains/subdomains. We then employed an in vitro dsRNA synthesis assay to test whether genotype R1, R2, R4, and R7 VP1 polymerases could be enzymatically activated by genotype C1, C2, C4, C5, and C7 VP2 core shell proteins. Genotype combinations that were incompatible informed the rational design and in vitro testing of chimeric mutant VP1 and VP2 proteins. The results of this study connect VP1 and VP2 nucleotide-level diversity to protein-level diversity for the first time, and they provide new insights into regions/residues critical for VP1-VP2 interaction(s) during viral genome replication.IMPORTANCE Group A rotaviruses (RVAs) are widespread in nature, infecting numerous mammalian and avian hosts and causing severe gastroenteritis in human children. RVAs are classified using a system that assigns a genotype to each viral gene according to its nucleotide sequence. To date, 22 genotypes have been described for the gene encoding the viral polymerase (VP1), and 20 genotypes have been described for the gene encoding the core shell protein (VP2). Here, we analyzed if/how the VP1 and VP2 proteins encoded by the known RVA genotypes differ from each other in their sequences. We also used a biochemical approach to test whether the intergenotypic sequence differences influenced how VP1 and VP2 functionally engage each other to mediate RNA synthesis in a test tube. This work is important because it increases our understanding of RVA protein-level diversity and raises new ideas about the VP1-VP2 binding interface(s) that is important for viral replication.

Dataset Information

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Publications

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

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