CNS-specific HIV reservoir characterization in humans: advancing our understanding through novel genomics methods.

The persistence of the human immunodeficiency virus (HIV) reservoir despite years of suppressive antiretroviral therapy (ART) represents the principal obstacle to achieving a functional cure. Because the intact, replication-competent portion of the reservoir drives viral rebound after treatment interruption, HIV cure strategies must specifically identify and target cells harboring these proviruses. Conventional reservoir‐characterization techniques typically require separate assays to capture integration sites, assess proviral integrity, and quantify clonal proliferation and they rely on short‐read sequencing. These limitations hinder our ability to understand how integration landscapes, the integrity of the HIV genome, and cell‐division dynamics intersect to maintain viral persistence in diverse tissues. We developed HIV SMRTcap, a single‐molecule, real‐time capture method that unifies long‐read sequencing with a customized bioinformatics pipeline. The assay employs targeted enrichment of HIV proviral DNA, followed by long read sequencing to recover contiguous proviral genomes and their flanking host sequences. We first validated SMRTcap’s sensitivity and performance across subtypes A, B, C, D, and A/D recombinants. We then applied the method to postmortem tissues—frontal lobe and spleen—from ART-suppressed, viremic, and HIV-associated encephalitis (HIVE) donors vii to characterize the HIV reservoir in tissues during different stages of infection. HIV SMRTcap effectively captured comprehensive reservoir characteristics across multiple HIV subtypes and tissue types. In the frontal lobe of ART-suppressed individuals, proviral integration predominantly found in intergenic regions, consisting of heavily truncated proviruses. In the other hand, the encephalitic donors exhibited variable cellular reservoir characteristics, reflecting different representation of disease severity. Notably, proviral populations in brain and spleen tissues primarily consisted of unique integration events or small clones, suggesting minimal clonal expansion. Integration site distributions varied among the groups but showed no preferential integration into zinc finger genes; however, these integrations persisted post-viral suppression. Together HIV SMRTcap is a sensitive, robust, and comprehensive tool capable of characterizing HIV reservoirs across various subtypes and tissues. Despite its current methodological limitations—such as distinguishing episomal from sheared genomic DNA and capturing hypermutated sequences—the assay significantly advances the understanding of tissue-specific HIV reservoirs. Further research with expanded cohorts and improved bioinformatic analysis will enhance reservoir characterization, guiding future HIV cure strategies.