Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

Abstract

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Author Summary

Bluetongue virus (BTV) is an economically important pathogen of ruminants that belongs to a group of viruses whose genome consists of multiple segments of double-stranded RNA. In order for the virus to synthesize viable and infectious progeny, a precise set of the 10 newly replicated BTV segments must be selected for packaging into each new virus particle. How the virus is able to select its own genomic strands from the vast array of cellular RNAs is not clearly understood. One possibility is that that BTV segments harbours an interaction signal that allows them to be sorted and packaged as a set. Correct identification of these signals has basic and applied implications for a possible target of antiviral therapeutics through inhibition of genome sorting and packaging process. Here we showed that a series of short oligonucleotides (ORNs) complementary to multiple sites on the BTV RNA prevented the growth of viable virus in infected cells. ORNs positive for inhibition in virus growth also prevented the genomic RNA to be packaged in an in vitro packaging assay. Moreover, when these same targeted sequences were deleted or mutated in viral genome, viable virus recovery was abolished. Exchanging the terminal sequences between segments failed to recover virus confirming that such changes are deleterious to virus viability. These studies have identified specific regions and sequences key to genome packaging in dsRNA viruses and viability. The specific genome packaging sequences targeted by inhibitory activities of ORNs are bona fide drug target which, as a mechanism common amongst all serotypes, may represent an Achilles’ heel for the development of virus therapeutics.