Progressive transformation of the HIV-1 reservoir cell profile over two decades of antiviral therapy

Abstract

HIV-1 establishes a life-long reservoir of virally infected cells which cannot be eliminated by antiretroviral therapy (ART). Here, we demonstrate a markedly altered viral reservoir profile of long-term ART-treated individuals, characterized by large clones of intact proviruses preferentially integrated in heterochromatin locations, most prominently in centromeric satellite/micro-satellite DNA. Longitudinal evaluations suggested that this specific reservoir configuration results from selection processes that promote the persistence of intact proviruses in repressive chromatin positions, while proviruses in permissive chromosomal locations are more likely to be eliminated. A bias toward chromosomal integration sites in heterochromatin locations was also observed for intact proviruses in study participants who maintained viral control after discontinuation of antiretroviral therapy. Together, these results raise the possibility that antiviral selection mechanisms during long-term ART may induce an HIV-1 reservoir structure with features of deep latency and, possibly, more limited abilities to drive rebound viremia upon treatment interruptions.

Keywords: FLIP-seq; HIV; HIV cure; HIV eradication; MIP-seq; block and lock; integration sites; latency; post-treatment controllers; retroviral pathogenesis.

Graphical abstract

Figure 1

Proviral reservoir profile in long-term ART-treated individuals (A and B) Frequency of total (A) and intact (B) HIV-1 proviruses in long-term ART-treated individuals (LT-ART), in people living with HIV (PLHIV) undergoing moderate durations of treatment (median of 9 years) (m-ART), and in elite controllers (ECs). Open circles indicate data at the limit of detection. (C) Proportion of intact proviruses within total HIV-1 proviruses in indicated study cohorts. (D) Proportion of proviruses with genome-intact or defective sequences in indicated study cohorts. (E) Average genetic distance between intact proviruses from indicated study cohorts, determined by pairwise comparisons between all intact proviruses from a given study participant. (F) Pie charts reflecting proportions of intact proviruses detected once (non-clonal) or multiple times (clonal) in the three indicated study cohorts. (G) Proportions of clonally expanded intact HIV-1 proviruses within the total pool of intact HIV-1-proviruses from each study participant. (H) (Left panel) Proportion of wild-type clade B CTL epitopes restricted by autologous HLA class I alleles within intact proviruses from indicated study cohorts. Each symbol represents one intact proviral sequence; all intact clade B sequences were included. (Right panel) Numbers of base pair variations significantly associated with autologous HLA class I alleles, determined as described by Carlson et al., within intact HIV-1 proviruses from indicated study participants. Each symbol reflects one intact provirus. Clonal sequences were counted once. Box and Whisker plots demonstrate median, interquartile ranges, and minimum/maximum. (A–H) FDR-adjusted two-sided Kruskal-Wallis nonparametric test or FDR-adjusted Fisher’s exact test were used, as appropriate. (A–C, E, and G) Horizontal bars indicate the median and n represents the number of study subjects. (D, F, and H) n reflects the number of viral sequences.

Figure 2

Chromosomal positioning of intact HIV-1 proviruses in long-term ART-treated individuals (A) Maximum-likelihood phylogenetic trees for intact HIV-1 proviruses from long-term ART-treated individuals (LT01–LT08). Coordinates of chromosomal integration sites obtained by integration site loop amplification (ISLA) and corresponding gene name (if applicable) are indicated. Symbols indicate sequences generated by FLIP-seq, MIP-seq, PRIP-seq or from quantitative viral outgrowth assays (qVOAs). Proviruses integrated in highly repetitive satellite DNA could not always be definitively mapped to specific chromosomal locations; a detailed list of integration sites is shown in Table S1. ^∗Sequences differ by 1 or 2 base pairs from adjacent clonal sequences. LADs, lamina-associated domains. (B) Proportions of intact proviruses with indicated integration site features in LT-ART individuals and comparison cohorts. (C) Chromosomal distance between integration sites of intact proviruses to most proximal host transcriptional start sites (TSSs), as determined by RNA-seq in CD4 T cells from reference datasets in total, effector-memory (EM), or central-memory (CM) primary CD4 T cells or from genome browser (GB). Box and Whisker plots show median, interquartile ranges and minimum/maximum. (D) Proportions of genome-intact proviral sequences in structural compartments/subcompartments A and B, as determined by Hi-C seq data. Integration sites not covered in the reference dataset were excluded. (B–D) Data from individuals with moderate ART treatment durations (m-ART) and from EC are shown for comparison. Clonal sequences are counted once. (B–D) p values were calculated by FDR-adjusted two-sided Kruskal-Wallis nonparametric tests or chi-square tests, adjusted for multiple comparison testing where applicable. n reflects the number of integration sites.

Figure 3

Transcriptional activity of single HIV-1 proviruses from study participant LT03 (A) Maximum-likelihood phylogenetic tree of individual proviruses isolated from LT03 using PRIP-seq. Chromosomal integration sites are indicated where available; genes harboring the integration site are shown where applicable. Color coding reflects the transcriptional activity of proviral species. (B) Genome browser snapshot indicating RNA-seq, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and ChIP-seq reads surrounding the chromosomal integration site of the indicated proviral sequence from study participant LT03; RNA-seq and ATAC-seq reads are derived from reference data of HIV-1-infected ART-treated persons and were first described in Einkauf et al.; ChIP-seq reads from the reference dataset of the ROADMAP consortium were used.

Figure 4

Longitudinal evolution of intact proviral reservoir landscape during long-term antiretroviral therapy (A) CIRCOS plots reflecting the chromosomal locations of intact proviruses at indicated time points in five study participants (LT01–LT04, LT08). Each symbol reflects one intact provirus. Clonal sequences, defined by identical integration sites and/or complete sequence identity, are highlighted. (B) Pie charts reflecting longitudinal evolution of chromosomal integration site landscapes of intact proviruses at indicated time points. Data indicate proportions of intact proviruses in indicated chromosomal locations. (C) Chromosomal distance between integration sites of intact proviruses to most proximal host TSS, as determined by RNA-seq in CD4 T cells from reference datasets in total, effector-memory (EM), or central-memory (CM) primary CD4 T cells or from genome browser (GB). Box and Whisker plots show median, interquartile ranges and minimum/maximum. (D) Proportions of genome-intact proviral sequences in structural compartments/subcompartments A and B, as determined by Hi-C seq data. Integration sites not covered in the reference dataset were excluded. For T3, data from 2017 and 2020 from LT01 were pooled. (B–D) Clonal sequences are counted once. n reflects the number of integration sites. p values were calculated by chi-square tests in (B) and (D), and by FDR-adjusted two-sided Kruskal-Wallis nonparametric tests in (C), adjusted for multiple comparison testing where applicable.

Figure 5

Proviral landscape of defective HIV-1 proviruses in long-term ART-treated individuals (A) CIRCOS plots reflecting the chromosomal locations of defective proviruses at indicated time points in five study participants (LT01–LT04, LT08). Each symbol reflects one defective provirus. Clonal sequences, defined by integration sites and/or complete sequence identity, are highlighted. Color-coded arches around the plots indicate types of defects in HIV-1 genomes. (B) Proportions of intact and defective proviruses with indicated integration site features at time points T1–T3. (C) Chromosomal distance between integration sites of intact and defective proviruses to most proximal TSS, as determined by RNA-seq in CD4 T cells from reference datasets at indicated time points. Box and Whisker plots show median, interquartile ranges and minimum/maximum. (B and C) Data from defective proviruses after 20 years of suppressive antiretroviral therapy are cross-sectionally compared with corresponding data from intact proviruses. A complete list of defective proviruses and their corresponding chromosomal location is indicated in Table S1. p values were calculated by a chi-square test in (B) and by an FDR-adjusted two-sided Kruskal-Wallis nonparametric test in (C), adjusted for multiple comparison testing where applicable. n reflects the number of integration sites.

Figure 6

Longitudinal dynamics of intact proviruses in two individuals with post-treatment control (A) Longitudinal analysis of HIV-1 plasma viral load in study persons 04 and 30. PBMC sampling time points are indicated by arrows. Day 0 is the first day of treatment interruption. (B) CIRCOS plots indicating longitudinal evolution of intact proviruses and their corresponding chromosomal integration sites. Each symbol reflects one intact provirus. Clonal sequences, defined by identical integration sites and/or complete sequence identity, are highlighted. In study person 30, two clones were detected in repetitive genomic regions in immediate proximity to micro-satellite DNA; due to the repetitive nature of these regions, the exact chromosomal region could not be definitively identified. ^∗Intact proviral sequences analyzed without identification of integration sites that differ by 1 or 2 base pairs from adjacent clonal sequences and might be part of the respective clones. (C) CIRCOS plots indicating longitudinal evolution and chromosomal locations of defective HIV-1 proviruses in study persons 04 and 30. Color-coded arches around the plots indicate types of defects in HIV-1 genomes.

Figure 7

Integration site profile of intact proviruses from study persons with virological rebound after ART interruption (A) Longitudinal analysis of HIV-1 plasma viral load in three study persons who developed rapid viral rebound following ART interruption. PBMC sampling time points are indicated by arrows. (B) Maximum likelihood phylogenetic tree for intact proviruses isolated from PBMC samples prior to ART interruption in three rebounders shown in (A). Chromosomal integration sites are indicated when available. (C) Proportions of intact and defective proviruses with indicated integration sites in PTC. Data from intact proviruses identified in rebounders are shown for comparison. Data from the time point immediately prior to treatment interruption are shown; clonal sequences are counted individually. Significance was calculated using a chi-square test, adjusted for multiple comparison testing. n reflects the number of integration sites.