Speaker
Description
HIV-1 persistence during antiretroviral therapy (ART) remains the main barrier to a cure. Eliminating these reservoirs is extremely challenging, in part because HIV-1 exhibits extraordinary genetic variability, generating heterogeneous populations capable of drug resistance and immune evasion. Although ART dramatically reduces viral population size, reservoirs retain sufficient genetic diversity to enable rapid rebound after therapy interruption, even after years of effective viral suppression. This residual diversity supports viral fitness recovery following population contraction, facilitating successful rebound. However, the genetic and evolutionary features permitting long-term persistence and rebound remain poorly defined.
HIV-1 populations are best described as viral quasispecies – a swarm of closely related variants or haplotypes. Long-read deep-sequencing of host-integrated proviral DNA and viral RNA allows quasispecies reconstruction by capturing longer stretches of viral genomes and preserving per-haplotype information inaccessible with short-read sequencing. However, long-read sequencing is prone to higher error rates, complicating haplotype reconstruction by confounding technical errors with true biological variation. Existing computational tools are optimized for short-read sequencing, assume short-read-specific error profiles, are computationally expensive, and/or underutilize the haplotype resolution provided by long reads.
To address these limitations, we developed OPOSSUM (Optimized Polishing of Observed reads for Strain Separation with Unsupervised Methods), a method for viral quasispecies reconstruction from long-read sequencing. OPOSSUM integrates robust quality control, dimensionality reduction, and unsupervised clustering to generate refined haplotype sequences while maximizing long-read resolution. We validated OPOSSUM using long-read sequencing of defined mixtures of HIV-1 plasmids and single-genome HIV-1 from clinical samples. OPOSSUM accurately reconstructed haplotypes, correctly estimated their abundance, and identified true mutations from noise. We then applied OPOSSUM to proviral DNA from SIV-infected macaques, enabling reconstruction of intra-host viral dynamics during ART and post-ART rebound. OPOSSUM provides a framework to infer reservoir dynamics and guide targeted cure strategies toward the cell populations and tissue reservoirs responsible for rebound.
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