Ordered release of genomic RNA during icosahedral virus disassembly

Many icosahedral viruses release their genomes through a series of ordered conformational changes involving distinct disassembly intermediates. While previous studies have focused on rearrangements of the capsid protein shell, it remains unclear whether viral genomes undergo defined structural transitions or are released in a specific order. Here, we developed "PT-ClickSeq," a next-generation sequencing platform that natively profiles viral nucleic acids exposed during particle disassembly without RNA extraction or fragmentation. Using Flock House virus (FHV) as a model, which forms two well-defined disassembly intermediates, we found that FHV releases its RNA genome in a conserved, ordered manner: the 5' and 3' termini and specific internal loci are exposed first. Distinct genomic regions exhibited different energy barriers to release, suggesting a programmed exposure process. Complementary "vPAR-CL" analysis revealed progressive loss of RNA-capsid interactions, with RNA-protein interactions anti-correlated with genome release. Together, these findings show that the encapsidated genome actively orchestrates viral disassembly rather than serving as a passive cargo.IMPORTANCEViruses need to strike a balance between structural rigidity and flexibility to achieve both sufficient protection and rapid release of packaged genome into host cells. During the process of genome delivery, many viruses undergo a programmed disassembly process through successive morphological changes, which give rise to partially disassembled virus particles, termed disassembly intermediates. It is important to study these intermediates as "checkpoints" to understand virus disassembly dynamics. We established a next-generation sequencing method that can monitor the RNA behavior during these conformational changes. We found that different regions of RNA were released with different energy thresholds, and the RNA release prioritized regions with low RNA-protein interactions. These findings shed light on the active role of the viral RNA in virus disassembly.