Mapping RNA-capsid interactions and RNA secondary structure within authentic 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: Photo-Activatable Ribonucleoside Cross-Linking (PAR-CL). In PAR-CL, 4-thiouracil is incorporated into the encapsidated genomes of authentic virus particles and subsequently UV-crosslinked to adjacent capsid proteins. We demonstrate that PAR-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 PAR-CL signals across virus RNA genome indicating a clear tropism of the encapsidated RNA genome. Certain interaction sites correlate to previously identified FHV 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 PAR-CL and DMS-MaPseq reveals that the predominant RNA-capsid sites favor double-stranded RNA regions. We disrupted secondary structures associated with PAR-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.