Transcription of HIV-1 at sites of intact latent provirus integration

Abstract

HIV-1 anti-retroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here we report on 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. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 10010,000X less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir thereby influencing cytopathic effects and proviral immune evasion.

Figure 1.. Selected integration sites and production of reporter T cell clones.

(A) Table of selected integration sites. (B) Schematic representation of the methods used to produce reporter T cell lines (see Materials and methods section for details). Created with BioRender.com.

Figure 2.. Jurkat reporter lines.

(A) Histograms show GFP fluorescence (x-axis) per normalized counts (y-axis) for each integration site studied (iStrains, red) and control (blue), in both resting (upper panel) and PMA/ionomycin-activated (lower panel) conditions. (B) Graphs show relative LTR (left panel), eGFP (middle panel) and nLuciferase (right panel) expression assessed by qPCR under resting (blue) and PMA/ionomycin-activated (red) conditions. Bars represent the mean relative expression from two independent assays (biological replicates) ± standard deviation. (C) LTR transcripts per cell (y-axis), determined by qPCR, for each integration-positive clone and control, under resting (blue) and activated (red) conditions. CD4⁺ T cells infected with HIV-1_Yu2 served as positive control (black bar). Bars represent the mean of two independent assays (biological replicates) ± standard deviation. (D) Relative expression determined by qPCR for host genes neighboring their respective reporter proviruses (iStrains, full bars) and control (stripped bars), under resting (left panel, blue) and PMA/ionomycin-activated (right panel, red) conditions. Bars represent the mean relative expression of two independent assays (biological replicates) ± standard deviation. Expression of host gene was averaged across multiple clones to the same reporter integration site. (E) Correlation between the relative expression of LTR (y-axis) for each Jurkat clone and their respective host gene (x-axis), averaged across multiple clones to the same reporter integration site, under resting (left panel, blue) and PMA/ionomycin-activated (right panel, red) conditions. Person’s correlation coefficients, r, and two-tailed p values were computed for each condition.

Figure 3.. Primary CD4⁺ T cell reporter lines.

(A) Histograms show GFP fluorescence (x-axis) per normalized counts (y-axis) for each integration site studied (iStrains, red) and control (blue), in both resting (upper panel) and CD3/CD28-activated (lower panel) conditions. (B) Graphs show relative LTR (left panel), eGFP (middle panel) and nLuciferase (right panel) expression assessed by qPCR under resting (blue) and CD3/CD28-activated (red) conditions. Bars represent the mean relative expression from two independent assays (biological replicates) ± standard deviation. (C) LTR transcripts per cell (y-axis), determined by qPCR, for each integration-positive clone and control, under resting (blue) and CD3/CD28-activated (red) conditions. CD4⁺ T cells infected with HIV-1_Yu2 served as positive control (black bar). Bars represent the mean of two independent assays (biological replicates) ± standard deviation. (D) Relative expression determined by qPCR for host genes neighboring their respective reporter proviruses (iStrains, full bars) and control (stripped bars), under resting (left panel, blue) and CD3/CD28-activated (right panel, red) conditions. Bars represent the mean relative expression of two independent assays (biological replicates) ± standard deviation. (E) Correlation between the relative expression of LTR (y-axis) for each primary T cell clone and their respective host gene (x-axis), averaged across multiple clones to the same reporter integration site, under resting (left panel, blue) and activated (right panel, red) conditions. Person’s correlation coefficients, r, and two-tailed p values were computed for each condition.

Figure 4.. Chromatin accessibility

(A-B) Chromatin accessibility in Jurkat (A) and primary CD4⁺ T cell clones (B) as measured by ATAC-seq for the reporter construct at each integration site and control under resting conditions (C) Graph shows ATAC-seq for ATP2B4 encompassing the reporter integration site in control, and clones that carry the reporter in KDM2A, ATP2B4. Blue shading indicates the site of reporter integration. Graphs were generated by averaging the normalized reads from three technical replicates for each clone.

Figure 5.. HIV-infected cells from people living with HIV.

(A) Schematic representation of the methods used to grow out HIV-1 infected cells from ART suppressed individuals (Weymar et al., 2022). Created with BioRender.com. (B-C) Uniform manifold approximation and projection (UMAP) of 10X single cell gene-expression data showing the position of the cells expressing the latent clones’ specific TCR (red dots) (B) and the fraction of latent cells in each cluster (C), for participants 603 (upper panels) and 5104 (lower panels) from ex vivo cells (left panels, data from Weymar et al., 2022), and cultured cells under resting (middle panels) and activated (right panels) conditions. (D) Histograms show HIV-1 Gag p24 expression in HIV+ cells (red) and non-infected cells (blue) of cultures derived from 603 and 5104, under resting (upper panel) and activated (lower panel) conditions. (E) Relative expression of LTR (purple bars), Gag (red bars) and Env (blue bars) by qPCR for 603 and 5104 HIV+ cells under resting and activated conditions. Bars represent the mean relative expression of two independent assays (biological replicates) ± standard deviation. (F) LTR transcripts per cell determined by qPCR, in cells from 603 and 5104 and in Jurkat and primary T cells reporter lines with proviruses integrated into iATP2B4, under resting (blue) and activated (red) conditions and HIV-1_YU2 controls. Bars represent the mean of two independent experiments (biological replicates) ± standard deviation. (G) Relative expression determined by 10X Genomics single cell mRNA sequencing of host genes neighboring HIV-1 proviral integration (ZNF486, participant 603 and ATP2B4, participant 5104) in HIV-infected (HIV+, full bars) and non-infected (HIV−, stripped bars) cells from the same cell population, under resting (blue bars) and activated (red bars) conditions. Bars represent the mean of the respective population from one assay ± standard deviation representing population variance.