How multi-segmented double-stranded RNA (dsRNA) viruses correctly incorporate their genomes into their capsids remains unclear for many viruses, including Bluetongue virus (BTV), a Reoviridae member, with a genome of 10 segments. To address this, we used an RNA-cross-linking and peptide-fingerprinting assay (RCAP) to identify RNA binding sites of the inner capsid protein VP3, the viral polymerase VP1 and the capping enzyme VP4. Using a combination of mutagenesis, reverse genetics, recombinant proteins and in vitro assembly, we validated the importance of these regions in virus infectivity. Further, to identify which RNA segments and sequences interact with these proteins, we used viral photo-activatable ribonucleoside crosslinking (vPAR-CL) which revealed that the larger RNA segments (S1-S4) and the smallest segment (S10) have more interactions with viral proteins than the other smaller segments. Additionally, using a sequence enrichment analysis we identified an RNA motif of nine bases that is shared by the larger segments. The importance of this motif for virus replication was confirmed by mutagenesis followed by virus recovery. We further demonstrated that these approaches could be applied to a related Reoviridae member, rotavirus (RV), which has human epidemic impact, offering the possibility of novel intervention strategies for a human pathogen.
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.