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.
Highlights
Avian reoviruses (ARVs) are commercially significant pathogens that cause considerable losses in the poultry industry worldwide [1]
Our results show that the ARV NS is capable of accelerating RNA folding, functioning as an RNA chaperone [15], consistent with its role in genomic segment precursor selection by facilitating specific RNA–RNA interactions in viroplasms
Its early elution from the size-exclusion column suggests that NS oligomerizes, whilst circular dichroism spectroscopy confirms that the protein is folded, and composed of ∼35% -strands and ∼29% ␣-helices (Supplementary Figure S1)
Summary
Avian reoviruses (ARVs) are commercially significant pathogens that cause considerable losses in the poultry industry worldwide [1]. ARVs belong to the genus Orthoreovirus of the Reoviridae family, which encompasses numerous human and animal pathogens, including rotaviruses, bluetongue virus and coltiviruses. Reoviruses possess double-stranded (ds) RNA genomes, partitioned into 10–12 genomic segments [2]. The ARV genome (23.5 kb) is composed of 10 dsRNA segments, encoding 8 structural and at least four nonstructural proteins [1]. The genomic dsRNA segments are encased within two concentric protein shells, forming the outer capsid and the core. Apart from genomic segments the core contains multiple copies of the RNA-dependent RNA polymerase and the capping enzyme [2]. Upon infection the outer capsid is proteolytically removed, resulting in the transcriptional activation of the core. The released core extrudes mRNAs into cytoplasm [3], where they are used for both translation and as genome segment precursors
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