Rotavirus Genomic RNA Complex Forms via Specific RNA–RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity

Abstract

Rotavirus (RV), a member of the Reoviridae family, causes infection in children and infants, with high morbidity and mortality. To be viable, the virus particle must package a set of eleven RNA segments. In order to understand the packaging mechanism, here, we co-synthesized sets of RNA segments in vitro in different combinations and detected by two alternate methods: the electrophoretic mobility shift assay (EMSA) and the RNA-bead pull-down assay. We showed that viral positive-sense RNA segments interact with each other in a specific manner, forming RNA complexes, and that the RNA–RNA interactions followed a sequential order initiated by small RV segments. Further, we demonstrated that RNA complexes were perturbed by targeted specific antisense oligoribonucleotides (ORNs) complementary to short RNA sequences, indicating that the RNA–RNA interactions between different segments were sequence-specific. The same inhibitory ORNs also had the capability to inhibit virus replication. The combined in vitro and in vivo data inferred that RNA–RNA interactions and specific complex formation are essential for sorting different segments, possibly prior to, or during, genome packaging. As genome assembly is a universal requirement in the Reoviridae family, this work offers an approach towards a further understanding of the sorting and packaging mechanisms of RV and related dsRNA (double-stranded RNA) viruses.

Rotavirus: Genetics, pathogenesis and vaccine advances

“…In channel generality model, multiple transcripts of gene segments can be transported out of pores on the viral capsid, and one segment at a time can extrude out of the capsid . The plus‐stranded RNAs can serve as a template for replication and translation of dsRNA segments (progeny segments) and virus encoded proteins, respectively …”

“…82,83 The plus-stranded RNAs can serve as a template for replication and translation of dsRNA segments (progeny segments) and virus encoded proteins, respectively. 84…”

Dynamic network approach for the modelling of genomic sub-complexes in multi-segmented viruses

“…To validate the mathematical model of RNA complexes between S6–S10, we used an established in vitro RNA–RNA interaction assay (9,12). Each RNA transcript was synthesized individually from T7 plasmids, purified and the molecular size of each was confirmed by denaturing agarose gel electrophoresis (Figure 2A, panel i).…”

Trans-Acting RNA–RNA Interactions in Segmented RNA Viruses

“…The genus Rotavirus is in the same Sedoreovirinae subfamily as orbiviruses, but these viruses contain eleven dsRNA genome segments. Similar to BTV, in vitro studies on rotaviruses (RVs) support the involvement of mRNA tRRI networks in segment packaging [39,40], however these tRRIs still require functional validation in virus infections. Interestingly, in vitro analysis of RV segment interactions revealed that virally-encoded NSP2, an RNA-binding protein, is able to remodel RNA segments in a manner that enhances tRRIs [40].…”