Transcription of HIV-1 at sites of intact latent provirus integration [scRNA-seq]

The study examined proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines and showed that the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir Overall design: CD4+ T cells harboring intact latent HIV-1 proviruses were enriched using their T cell receptors (Weymar et al., 2022). CD4+ T cells from participants 603 (Rockefeller University IRB-approved Protocols MCA-0866/TSC-0910) and 5104 (Protocols MCA-0965/TSC-0910) were isolated by magnetic separation using CD4+ T Cell Isolation Kit, human (Miltenyi Biotec, cat. 130-091-155) and CD45RA MicroBeads, human (Miltenyi Biotec, cat. 130-045-901) and prepared for cell sorting exactly as described (Weymar et al., 2022). Cells from 603 (CD4+TRBC1+TRBV19+) and 5104 (CD3+CD4+TRBC1+TRBV) were sorted into 96 well “U” bottom plates (5 cells per well) containing 200 µL of activation media (composition described above), plus feeder cells, supplemented with the following ARVs: 1µM Tenofovir, 1µM Emtricitabine, 1nM Nevirapine and 10 µM T-20. Cell sorting was performed on FACSymphony S6 using FACSDiva software (version 9.5.1, BD Biosciences) and data was analyzed using FlowJo (version 10.10.0, BD Biosciences). The culture media was replaced twice a week to replenish ARVs and avoid new infections. Three weeks after sorting, cells were screened for the presence of the HIV clone of interest by Env PCR and sequencing (Salazar-Gonzalez et al., 2008). Cell lines containing clones of interest were expanded in a T25 culture flask in 10 mL of activation medium. The frequency of latent cells in the cultures was determined three weeks after expansion by sorting single cells into 10 µL of RLT buffer (Qiagen, cat 79216) using Agencourt RNAClean XP and magnetic beads (Beckman Coulter, cat. A63987) for DNA preparation and Env PCR.