Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing

To characterize RNA–capsid binding sites genome-wide within mature RNA virus particles, we have developed a Next-Generation Sequencing (NGS) platform: viral Photo-Activatable Ribonucleoside CrossLinking (vPAR-CL). In vPAR-CL, 4-thiouridine is incorporated into the encapsidated genomes of virus particles and subsequently UV-crosslinked to adjacent capsid proteins. We demonstrate that vPAR-CL can readily and reliably identify capsid binding sites in genomic viral RNA by detecting crosslink-specific uridine to cytidine transitions in NGS data. Using Flock House virus (FHV) as a model system, we identified highly consistent and significant vPAR-CL signals across virus RNA genome, indicating a clear tropism of the encapsidated RNA genome. Certain interaction sites coincide with previously identified functional RNA motifs. We additionally performed dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to generate a high-resolution profile of single-stranded genomic RNA inside viral particles. Combining vPAR-CL and DMS-MaPseq reveals that the predominant RNA–capsid interaction sites favored double-stranded RNA regions. We disrupted secondary structures associated with vPAR-CL sites using synonymous mutations, resulting in varied effects to virus replication, propagation and packaging. Certain mutations showed substantial deficiency in virus replication, suggesting these RNA–capsid sites are multifunctional. These provide further evidence to support that FHV packaging and replication are highly coordinated and inter-dependent events.