Tracking HIV-1 DNA fate from cell culture to humanized mice tissues

Unambiguous identification of HIV reservoirs is essential for their characterization and for developing curative strategies. A major obstacle to curing HIV is the persistence of silent viral genomes in host cells that evade immune detection and resist therapy. Here, we present a fluorescence microscopy-based method for direct in vivo visualization of full-length double-stranded HIV-1 DNA, an achievement not previously realized. To achieve this, we developed an advanced imaging platform adapted for replication-competent HIV-1. This system leverages a bacterial-derived two-component tagging strategy, where a fluorescent protein (OR-GFP), efficiently expressed in xenografted human immune cells, specifically binds to an engineered ANCH3 tag sequence integrated into the viral genome. The tagged virus infects CD4+ T cells both in vitro and in vivo, producing bright nuclear puncta corresponding to individual viral genomes. Multiple nucleation sites within ANCH3 enable a stable, shortened tag form for persistent and long-term tracking. Transcriptional profiling during acute infection revealed both transcriptionally active and silent genomes in the spleen, lymph nodes, and bone marrow, with silent forms enriched in lymph nodes and marrow, supporting early reservoir establishment. This live-cell imaging strategy enables high-specificity detection of latent HIV, offering a transformative tool for studying reservoir dynamics and guiding future cure strategies.