Mapping HIV-1 RNA structure, homodimers, long-range interactions and persistent domains by HiCapR.

Human Immunodeficiency Virus type 1 (HIV-1) RNA genome organization remains a critical knowledge gap in understanding its replication cycle. To address this, we developed HiCapR, a psoralen crosslinking-based RNA proximity ligation method coupled with post-library hybridization, enabling high-resolution mapping of RNA-RNA interactions across the HIV-1 genome. This approach confirmed canonical structural motifs, including stem-loop architectures in the 5'-untranslated region (5'-UTR) and Rev Response Element (RRE), as well as dimerization sites within the 5'-UTR critical for viral packaging. Notably, HiCapR identified novel homodimerization events distributed along the genome, suggesting an expanded regulatory role of RNA multimerization in splicing regulation and selective encapsidation. Intriguingly, while infected cells exhibited extensive long-range RNA interactions-particularly within the 5'-UTR-virion-packaged genomes displayed a marked reduction in such interactions, indicative of a structural transition from a loosely organized state to a condensed conformation. This spatial reorganization coincided with the preservation of stable genomic domains essential for dimerization, which persisted throughout virion assembly. These domains, enriched at homodimer interfaces, likely serve as structural scaffolds ensuring fidelity during genome packaging. This work establishes HiCapR as a robust tool for probing RNA interactomes and provides mechanistic insights into how HIV-1 exploits RNA topological heterogeneity to regulate its life cycle. The identification of conserved structural domains and transient interaction networks opens avenues for targeting RNA conformation in antiviral strategies.