Speaker
Description
During infection, human immunodeficiency virus (HIV) maintains a stably integrated reservoir of proviruses that persist within the host genome despite combined antiretroviral therapy (cART). Characterizing these reservoirs remains challenging due to insufficient phylogenetic resolution, particularly under cART, which limits our ability to assess proviral integration and replication competency accurately.
To overcome these limitations, we applied an optimized single-cell sequencing platform (scDNA-seq) to capture near-full-length (NFL) viral DNA, improving genome coverage and sequencing depth over gold standard, single-genome amplification and Sanger sequencing. Peripheral blood mononuclear cells (PBMCs) and lymph node (LN) suspensions were collected from three treatment-interrupted S[imian]IV animals prior to, during, and after cART. Targeted amplicons spanning the full SIV genome and circular junction for unintegrated long terminal repeat circles were sequenced. Results were validated using a five-probe digital droplet PCR (ddPCR) panel, achieving >92% accuracy in classifying defective proviruses.
In total from the three animals, we identified 1,127 unique SIV genomes achieving unprecedented throughput and resolution. Phylogenetic likelihood mapping revealed a 10-26% reduction in unresolved phylogenies for NFL genomes when compared to envelope glycoprotein (gp120) sequences alone, demonstrating the superior resolution by increased genome coverage. Importantly, inferred tissue origins of viral rebound exhibited a greater than two-fold increase in statistically supported branching events for NFL genomes relative to gp120. Tissue origins even differed between NFL and gp120 phylogenies, telling distinct stories regarding lymph node reservoir behavior.
Overall, the scDNA-seq approach significantly enhances phylogenetic resolution and accelerates the study of viral evolution and phyloanatomy, offering a powerful tool for investigating proviral reservoir dynamics. Incorporating broader sample types and time points could provide transformative insights into the maintenance and behavior of defective and intact proviruses, advancing our understanding of reservoir dynamics and HIV persistence.
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