Identification of Genetically Intact HIV-1 Proviruses in Specific CD4+ T Cells from Effectively Treated Participants

Abstract

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4⁺ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Keywords: HIV; antiretroviral therapy; cellular proliferation; clonal expansion; full-length HIV sequencing; latency; replication competency; single proviral sequencing.

Figure 1. Full-Length Individual Proviral Sequencing (FLIPS) assay

(A) Primer binding sites in HIV-1 5′ and 3′ LTR regions used by FLIPS to amplify full-length (defective and intact) HIV-1 proviruses via nested PCR. (B) Process used to identify genetically intact and defective de novo assembled proviruses. (C) Percentage of defective and intact proviruses within the total pool of sequenced proviruses. (D) Percentage of intact proviruses sequenced for each participant. (E) Percentage of PCR reactions positive for amplified product with increasing copies of pWT/BaL, data represented as mean ± SD (n=2).

Figure 2. Comparison of intact proviruses between cell subsets

(A) Number of intact proviruses per million cells in each cell subset. (B) Proportion of each cell subset to the total pool of CD4⁺ T cells (C) Contribution of each CD4⁺ T cell subset to the overall intact reservoir. Data represented as mean ± CI for all graphs. Confidence intervals are calculated from one sample t-tests, except for T_CM in panel A, whose confidence interval upper bound is calculated from the product of the mean proportion of HIV+ cells in the T_CM subset, and the upper bound of the confidence interval for the proportion of proviruses that are intact for the T_CM subset. See also Figure S1.

Figure 3. Representative phylogenetic tree of HIV-1 sequences from a participant on long-term ART

Participant 2026. Horizontal lines adjacent to tips represent the individual contig sequence aligned to HXB2. Red asterisks denote intact sequences; black box indicates identical intact sequences. See also Data S1A–E.

Figure 4. Comparison of FLIPS to other assays

(A) Frequency of cells containing either intact or integrated HIV-1 DNA. (B) Number of intact proviruses identified using the FLIPS assay compared to simulated SPS of gag-pol and env regions. Data represented as mean ± CI for all graphs.