Extended Data Fig. 6: Sequence analysis of Q2 proviruses. | Nature

Extended Data Fig. 6: Sequence analysis of Q2 proviruses.

From: A quantitative approach for measuring the reservoir of latent HIV-1 proviruses

Extended Data Fig. 6

a, Sorting of productively infected CD4+ T cells. Cell preparations with a high fraction of intact proviruses were obtained by infecting CD4+ T lymphoblasts with a replication-competent HIV-1 carrying GFP in the nef ORF (R7-GFP37). After 48 h, GFP+ cells were collected by sorting. Genomic DNA was isolated, subjected to pulse field electrophoresis to remove unintegrated intermediates, and analysed by IPDA. b, IPDA analysis of high molecular mass DNA from sorted cells. Droplets in Q1 and Q4 largely reflect the shearing of intact proviruses (DSI = 0.46) during in DNA isolation and purification. c, Frequency of intact proviruses in GFP and GFP+ cells before and after correction for shearing. After correction for shearing, the frequency of intact proviruses in sorted GFP+ cells is close to the expected value of 1. d, Map of the HIV-1 genome in GFP-expressing HIV-1 vector R7-GFP used in a. GFP is inserted in the nef ORF. Positions of outer primers in the LTR and GFP used in single genome amplifications are indicated. e, Sequence analysis of nine independent single genomes. Arrows indicate positions of the Ψ and env IPDA amplicons. Orange lines indicate intact sequence without deletions or hypermutation and identical to R7-GFP except for single base mutations (black lines).

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