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Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

ABSTRACT: Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA-RNA interactions between genomic precursors during segment assortment and packaging.

SUBMITTER: Borodavka A

PROVIDER: S-EPMC4538827 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

Borodavka Alexander A Ault James J Stockley Peter G PG Tuma Roman R

Nucleic acids research 20150624 14

Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist pac ...[more]

PMID: 26109354

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Project description:The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp) enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses.We determined the L2 genome segment nucleotide sequences, which encode the RdRp proteins, of two different avian reoviruses, strains ARV138 and ARV176 in order to define conserved and variable regions within reovirus RdRp proteins and to better delineate structure/function of this important enzyme. The ARV138 L2 genome segment was 3829 base pairs long, whereas the ARV176 L2 segment was 3830 nucleotides long. Both segments were predicted to encode lambdaB RdRp proteins 1259 amino acids in length. Alignments of these newly-determined ARV genome segments, and their corresponding proteins, were performed with all currently available homologous mammalian reovirus (MRV) and aquareovirus (AqRV) genome segment and protein sequences. There was approximately 55% amino acid identity between ARV lambdaB and MRV lambda3 proteins, making the RdRp protein the most highly conserved of currently known orthoreovirus proteins, and there was approximately 28% identity between ARV lambdaB and homologous MRV and AqRV RdRp proteins. Predictive structure/function mapping of identical and conserved residues within the known MRV lambda3 atomic structure indicated most identical amino acids and conservative substitutions were located near and within predicted catalytic domains and lining RdRp channels, whereas non-identical amino acids were generally located on the molecule's surfaces.The ARV lambdaB and MRV lambda3 proteins showed the highest ARV:MRV identity values (approximately 55%) amongst all currently known ARV and MRV proteins. This implies significant evolutionary constraints are placed on dsRNA RdRp molecules, particularly in regions comprising the canonical polymerase motifs and residues thought to interact directly with template and nascent mRNA. This may point the way to improved design of anti-viral agents specifically targeting this enzyme.

Dataset Information

Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

Publications

Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

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