HIV latency in isolated patient CD4+ T cells may be due to blocks in HIV transcriptional elongation, completion, and splicing

Abstract

Latently infected CD4⁺ T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4⁺ T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription. In addition, we measured the degree to which these transcriptional blocks could be reversed ex vivo by T cell activation (using anti-CD3/CD28 antibodies) or latency-reversing agents. We found that the main reversible block to HIV RNA transcription was not inhibition of transcriptional initiation but rather a series of blocks to proximal elongation, distal transcription/polyadenylation (completion), and multiple splicing. Cell dilution experiments suggested that these mechanisms operated in most of the HIV-infected CD4⁺ T cells examined. Latency-reversing agents exerted differential effects on the three blocks to HIV transcription, suggesting that these blocks may be governed by different mechanisms.

Fig. 1. HIV transcription profiles in freshly isolated CD4⁺ T cells and PBMCs from HIV-infected patients on ART suggest blocks to HIV transcriptional elongation, completion, and multiple splicing

(A to C) Total RNA from unstimulated [day 0 (d0)] CD4⁺ T cells (A and C) and PBMCs (B) was used for a polyadenylation-RT-ddPCR assay for the TAR loop (found in all HIV transcripts) and RT-ddPCR assays for HIV sequence regions suggesting transcriptional interference (read-through transcripts), transcriptional elongation (long LTR), completion of transcription (polyA), and multiple splicing (Tat-Rev). Each transcript was normalized to 1 μg of cellular RNA (~10⁶ cells) and plotted on a log scale. (C) To determine whether there are blocks to more distal elongation beyond the long LTR region, additional assays were developed for HIV Pol and Nef, and all seven transcripts were measured in six HIV-infected patients on ART. (D and E). To evaluate how deletions or hypermutations in the provirus could affect the measured HIV RNA quantities, HIV DNA was measured using the same ddPCR assays (except Tat-Rev and polyA, which are RNA-specific), and each HIV RNA was normalized to the corresponding HIV DNA (polyA was normalized to the read-through assay, which uses the same forward primer/probe). Bars indicate the median. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.

Fig. 2. Activation of freshly isolated CD4⁺ T cells from HIV-infected patients on ART results in successive increases in elongated, polyadenylated, and multiply spliced HIV transcripts

(A to J) Using RT-ddPCR, read-through (A and F), total (B and G), elongated (C and H), polyadenylated (D and I), and multiply spliced Tat-Rev (E and J) HIV transcripts were measured in unstimulated CD4⁺ T cells (day 0) and ex vivo activated CD4⁺ T cells (day 2) from 12 ART-suppressed individuals. (A to E) Effect of activation, as normalized by RNA mass. Measures of each HIV transcript were normalized to 1 μg of cellular RNA (~10⁶ unstimulated cells) to show how activation changes amounts of each HIV transcript in relation to global cellular transcription. (F to J) Effect of activation, as normalized by DNA mass. HIV RNA measures in the unstimulated and activated cells were also normalized to 10⁶ cells using the cell equivalents recovered in the extracted DNA (a surrogate for cell numbers in the activated cells; available for 11 of 12 participants). Each graph shows the change in HIV RNA from day 0 (unstimulated) to day 2 (activated) on a log scale; each color represents a separate individual; comparisons were performed using the Wilcoxon signed-rank test.

Fig. 3. Activation of freshly isolated CD4⁺ T cells from HIV-infected patients on ART selectively reverses baseline blocks to splicing, elongation, and completion

The effects of activation were also measured using the ratio of one HIV transcript to another, which is independent of normalization to cell numbers. (A to D) Ratios to total (TAR) transcripts. HIV transcript measures in the unstimulated (day 0) and activated (days 1 and 2) CD4⁺ T cells were normalized to TAR to express the proportion of all HIV transcripts that are read-through (A), elongated (B), polyadenylated (C), and multiply spliced Tat-Rev (D) transcripts. (E to G) Ratios to long LTR. HIV transcript measures were normalized to long LTR to express the proportion of elongated HIV transcripts that are read-through (E), polyadenylated (F), and multiply spliced Tat-Rev (G) transcripts. (H) Ratio to polyadenylated. The ratio of Tat-Rev to polyA was used to express the proportion of completed transcripts that get multiply spliced to produce two-exon Tat-Rev. (I) Proportion of HIV transcripts blocked at the stages of transcriptional elongation, completion, and multiple splicing in unstimulated (day 0) and activated (day 2) CD4⁺ T cells. Each color represents a separate individual; comparisons were performed using the Wilcoxon signed-rank test.

Fig. 4. The frequency of CD4⁺ T cells containing each HIV transcript reflects the levels of each HIV transcript in the bulk cell population

Unstimulated (day 0) CD4⁺ T cells from three ART-suppressed HIV-infected individuals were counted and subjected to serial, replicate (≤6) fivefold cell dilutions. For two of these participants, another aliquot of cells was activated for 2 days with anti-CD3/CD28 antibody-coated beads (antiretroviral drugs were used to prevent new infection) and then serially diluted. Read-through, TAR, long LTR, polyA, and Tat-Rev transcripts were measured in each dilution and replicate. The number of HIV-infected cells in each replicate was inferred by measuring HIV DNA in the bulk cells. (A and B) The frequencies of unstimulated total CD4⁺ T cells (A) and HIV-infected CD4⁺ T cells (B) containing each HIV transcript were calculated using the proportion of positive replicates at each cell number using the method of extreme limiting dilution analysis (74). Bars indicate the median. (C to F) Effect of activation on the frequency of total CD4⁺ T cells (C and E) and HIV-infected CD4⁺ T cells (D and F) containing each transcript. Blue columns, unstimulated (day 0); red columns, activated (day 2). Bars represent 5 to 95% confidence intervals. P values were calculated using the website for extreme limiting dilution analysis (74). (G) Proportion of cells in which HIV transcription was blocked at the stages of elongation, completion, and splicing (of cells with TAR, long LTR, and polyA, respectively). Bars indicate the median.

Fig. 5. Putative latency-reversing agents exert differential effects on the different blocks to HIV transcription

(A to E) Total CD4⁺ T cells from five ART-suppressed HIV-infected individuals (A to E) were divided into aliquots of 5 × 10⁶ cells and cultured for 24 hours with dimethyl sulfoxide (DMSO) (negative control), different latency-reversing agents, or anti-CD3/ CD28 antibody-coated beads. Latency-reversing agents were used at concentrations that can be obtained in the plasma or those used in previous ex vivo studies. After 24 hours, cells were harvested from each well and used to measure read-through (orange), total (pink), elongated (green), polyadenylated (blue), and multiply spliced Tat-Rev (red) HIV transcripts using RT-ddPCR. Amounts of each transcript were divided by those in the negative control (DMSO) well to show the fold change (y axis) in each transcript (different colors) for each latency-reversing agent or control on the x axis. Ingenol 3,20, ingenol 3,20- dibenzoate; PEP005, ingenol mebutate (ingenol-3-angelate).