Lenacapavir Resistance Surveillance in Full-Length HIV-1 Proviral DNA and Circulating RNA: Major RAMs Are Rare and Absent in Intact Proviral DNA

Background Lenacapavir (LEN) has made the HIV-1 capsid relevant for resistance surveillance, but p24 variation within gag remains less well characterized than the resistance landscape of pol. This limits the clinical interpretation of capsid variation, particularly when detected in biologically distinct compartments such as proviral DNA and circulating RNA. We aimed to characterize p24 amino acid variation in full-length proviral DNA and circulating HIV-1 RNA and to assess the occurrence of Stanford-defined major LEN resistance-associated mutations (LEN RAMs). Methods Complete HIV-1 sequences and metadata were retrieved from the Los Alamos National Laboratory HIV Sequence Database and classified by nucleic acid source as DNA or RNA. Proviral DNA underwent O’Doherty Lab-derived full-length proviral classification, hypermutation screening, and clone-aware deduplication using a TN93 distance threshold of 0.015. RNA was analyzed in source-specific branches, using all QC-passed sequences for global variability analyses and one representative sequence per donor-year for grouped LEN RAM analyses. The p24 region was extracted after HXB2-guided alignment, and only QC-passed p24 sequences were retained for downstream analyses. Results A total of 22,456 complete HIV-1 sequences were retrieved. After source-specific preprocessing and p24 quality control, 9,063 RNA sequences, 12,787 non-deduplicated DNA sequences, and 3,878 deduplicated DNA sequences were available for analysis; the donor-year RNA branch retained 4,367 representative sequences. Across both sources, p24 was highly conserved, but recurrent substitutions clustered at a limited number of positions and were often shared between proviral DNA and circulating RNA. Major LEN RAMs were rare. In subtype B, detectable major LEN RAM signal was confined to non-intact proviral DNA, where Q67H and M66I were observed at low frequency, whereas no major LEN RAMs were identified in intact proviral DNA. No predefined LEN-associated combination patterns were detected. Conclusions Major LEN RAMs were rare in both proviral DNA and circulating RNA and were absent in intact subtype B proviral DNA. Although p24 was highly conserved, recurrent substitutions followed non-random patterns across compartments. These findings support cautious interpretation of gag variation detected in proviral DNA alone and do not provide evidence to support routine baseline capsid resistance testing before LEN use where such testing is not otherwise indicated.