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Fig. 1 | Retrovirology

Fig. 1

From: Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda

Fig. 1

Overview of the EDITS assay. Blood samples were obtained from HIV-infected individuals in Cleveland, OH, USA and Kampala, Uganda. Peripheral blood mononuclear cells (PBMC) were isolated and CD4+ memory T cells purified, counted and purity verified by flow cytometry. One million CD4+ memory T cells were induced for 16 h with 10 μg/ml of the mitogen Concanavalin A (cell-associated spliced HIV-1 RNA). Total RNA was purified and used as template in a One-Step RT-PCR (external), with primers designed to bind to either side of the HIV-1 Env RNA splice junction, yielding a product of approximately 1.9 kb from the spliced HIV-1 env mRNA. A nested PCR amplification using barcoded primers produced a 369 bp fragment corresponding to vpu/env (HIV-1HXB2 position 6026 to 6394), which was purified, quantified, and deep sequenced (MiSeq Illumina). Reads were analyzed using the DEEPGEN™ Software Tool Suite [51] and converted into the equivalent number of cells harboring HIV-1 per 106 cells using a standard curve as described [32]. The second aliquots of one million CD4+ memory T cells incubated in cell medium alone were used to isolate DNA. External PCR reactions amplified a 584 bp fragment, which was used as template for the nested PCR reaction, library preparation, deep sequencing, and bioinformatics analysis as described for the EDITS assay

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