Near full-length HIV-1 provirus analysis for characterizing intactness and variability in the viral reservoir
Year: 2025
Type: dissertation
Abstract: Despite effective antiretroviral therapy, HIV-1 persists in cellular reservoirs throughout the body, necessitating lifelong treatment and contributing to HIV-1-associated neuropathologies in people living with HIV. Traditional reservoir assessment methods suffer from limited genomic coverage, inability to distinguish intact from defective proviruses, and inadequate sensitivity for tissue-specific analysis. This work describes development and application of a comprehensive near full-length (NFL) nanopore sequencing diagnostic pipeline to characterize HIV-1 proviral reservoirs across diverse anatomical compartments and evaluate CRISPR-based therapeutic interventions. Intact and defective HIV-1 proviral genomes were characterized from peripheral blood mononuclear cells (PBMCs) and challenging tissue samples including brain and spleen. Novel analytical approaches include Maximal Deletion Size Analysis (MDSA) for quantifying genome intactness, Regional Deletion Size Analysis (RDSA) for gene-level characterization, and machine learning-based co-receptor tropism prediction. Application to spleen and brain tissues revealed tissue-specific reservoir characteristics. Participants with mild cognitive-motor disorder demonstrated the highest levels of intact sequences, challenging traditional disease progression models. Tissue-specific gene region deletion patterns emerged, with spleen showing significantly higher intact tat2 and rev2 exons compared to brain regions. Most significantly, strong positive correlation between viral genome intactness and CXCR4 tropism (r=0.52, p=0.0053) suggests co-selection of viral fitness and co-receptor usage. A Guide Compatibility Assay (GCA) was also developed with this technique to predict CRISPR editing efficiency using pre-treatment viral sequence data, demonstrating 81% accuracy. This diagnostic pipeline addresses critical limitations in current HIV-1 reservoir research by providing comprehensive, tissue-adaptable methods for characterizing viral persistence, establishing progress in discerning success in HIV-1 cure research.
DOI: 10.17918/00011098
Language: English
Topic: HIV Research and Treatment
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4412086662