Stability of local secondary structure determines selectivity of viral RNA chaperones

Abstract

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA–RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA–RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Assembly intermediates of orthoreovirus captured in the cell

“…The far larger number of segments and the recent studies of genome segment selection in the Reoviridae indicate that the mechanism used to ensure encapsidation of a complete set of 10-genome segments includes RNA-RNA recognition between segments, possibly to form a concatamer of RNA which is then recognized for packaging 3,[25][26][27] . If there is indeed some similarity in the assembly process, with collapsed particles being transformed into an expanded state by the recruitment of the RNA genome then the empty virion-like particles we observe would be dead-end products that have failed to package the genome.…”

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

“…Coacervation of the viral RNA chaperone NSP2 35,38,70 associated with multiple viral transcripts would accelerate formation of inter-molecular RNA-RNA interactions between them. Several processes may contribute to stabilisation of sequence-specific inter-molecular RNA-RNA contacts 44,71 , while promoting intra-molecular duplex melting via interactions with multiple arginine side chains of NSP2 72 concentrated in the viroplasmic liquid phase 6 .…”

Genome packaging in multi-segmented dsRNA viruses: distinct mechanisms with similar outcomes

“…It has been shown that NSP2 may play a role in preventing premature initiation of replication of the packaged +RNAs [ 65 ]. Although NSP2 can promote strand-annealing reactions between non-cognate complementary sequences [ 66 ], it appears that the RNA assortment in rotaviruses may be primarily attained via sequence-specific RNA–RNA interactions [ 49 •• , 67 •• ]. Importantly, non-cognate RNAs are outcompeted by cognate rotavirus RNA transcripts in RNA–RNA in vitro interaction assays in the presence of NSP2 [ 49 •• ].…”