Dataset Information

Group A Rotavirus VP1 Polymerase and VP2 Core Shell Proteins: Intergenotypic Sequence Variation and In Vitro Functional Compatibility.

ABSTRACT: 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.

SUBMITTER: Steger CL

PROVIDER: S-EPMC6321916 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Group A Rotavirus VP1 Polymerase and VP2 Core Shell Proteins: Intergenotypic Sequence Variation and <i>In Vitro</i> Functional Compatibility.

Steger Courtney L CL Boudreaux Crystal E CE LaConte Leslie E LE Pease James B JB McDonald Sarah M SM

Journal of virology 20190104 2

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 ext ...[more]

PMID: 30355692

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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:One step of the life cycle common to all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of nonstructural protein 5 (NSP5) with the RV core shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, and I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as a dominant-negative element by disrupting the formation of either VLS or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV species A to H. Detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication.IMPORTANCE Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients, adults traveling to high-risk regions, and even for the livestock industry. Aside from structural and physiological divergences among RV species studied before now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV protein NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral.

Dataset Information

Group A Rotavirus VP1 Polymerase and VP2 Core Shell Proteins: Intergenotypic Sequence Variation and In Vitro Functional Compatibility.

Publications

Group A Rotavirus VP1 Polymerase and VP2 Core Shell Proteins: Intergenotypic Sequence Variation and <i>In Vitro</i> Functional Compatibility.

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