Gut and blood differ in constitutive blocks to HIV transcription, suggesting tissue-specific differences in the mechanisms that govern HIV latency

Abstract

Latently-infected CD4+ T cells are widely considered to be the major barrier to a cure for HIV. Much of our understanding of HIV latency comes from latency models and blood cells, but most HIV-infected cells reside in lymphoid tissues such as the gut. We hypothesized that tissue-specific environments may impact the mechanisms that govern HIV expression. To assess the degree to which different mechanisms inhibit HIV transcription in the gut and blood, we quantified HIV transcripts suggestive of transcriptional interference (U3-U5; "Read-through"), initiation (TAR), 5' elongation (R-U5-pre-Gag; "Long LTR"), distal transcription (Nef), completion (U3-polyA; "PolyA"), and multiple splicing (Tat-Rev) in matched peripheral blood mononuclear cells (PBMCs) and rectal biopsies, and matched FACS-sorted CD4+ T cells from blood and rectum, from two cohorts of ART-suppressed individuals. Like the PBMCs, rectal biopsies showed low levels of read-through transcripts (median = 23 copies/106 cells) and a gradient of total (679)>elongated(75)>Nef(16)>polyadenylated (11)>multiply-spliced HIV RNAs(<1) [p<0.05 for all], demonstrating blocks to HIV transcriptional elongation, completion, and splicing. Rectal CD4+ T cells showed a similar gradient of total>polyadenylated>multiply-spliced transcripts, but the ratio of total to elongated transcripts was 6-fold lower than in blood CD4+ T cells (P = 0.016), suggesting less of a block to HIV transcriptional elongation in rectal CD4+ T cells. Levels of total transcripts per provirus were significantly lower in rectal biopsies compared to PBMCs (median 3.5 vs. 15.4; P = 0.008) and in sorted CD4+ T cells from rectum compared to blood (median 2.7 vs. 31.8; P = 0.016). The lower levels of HIV transcriptional initiation and of most HIV transcripts per provirus in the rectum suggest that this site may be enriched for latently-infected cells, cells in which latency is maintained by different mechanisms, or cells in a "deeper" state of latency. These are important considerations for designing therapies that aim to disrupt HIV latency in all tissue compartments.

Fig 1. The HIV genome and the targets for transcription profiling assays.

This schematic shows the genetic organization of proviral HIV DNA and the HIV ‘transcription profiling’ assays targeting specific RNA sequence regions that provide insight into blocks to transcription. Some proposed mechanisms underlying the blocks to transcription initiation, elongation, and splicing are detailed.

Fig 2. HIV RNA levels and HIV RNA/DNA ratios reveal blocks to elongation, distal transcription, and multiple-splicing in PBMCs and intact gut biopsies.

Read-through, total (TAR), 5’ elongated (R-U5/pre-Gag; “Long LTR”), Nef, polyadenylated (PolyA), and multiply-spliced Tat-Rev (MS Tat-Rev) HIV RNAs were measured in (A) PBMCs; and (B) intact rectal biopsies (n = 9 ART-suppressed individuals). 2C-D: Levels of HIV transcription per provirus are lower in the gut than the blood. Levels of each HIV RNA were normalized to HIV DNA from the same sample as measured by (C) ddPCR for the corresponding DNA sequence region (PolyA was normalized to the Read-through assay, which employs the same forward primer/probe), except for MS Tat-Rev where there is no DNA equivalent; or (D) ddPCR for the Long LTR assay, which is present once in each intact provirus. Bars indicate the median. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.

Fig 3. Stability of HIV transcripts ex vivo.

Peripheral CD4+ T cells were isolated from an ART-suppressed individual and treated with the RNA polymerase II (RNA Pol II) inhibitors (A) Triptolide [100 nM] or (B) Actinomycin D [5 mg/mL] to arrest de novo cellular and viral transcription. HIV transcripts (Read-through, TAR, Long LTR, Nef, PolyA, and MS Tat-Rev) were quantified using RT-ddPCR from cells harvested at various time points post-treatment. Levels of each HIV RNA were expressed as a proportion of the level at time t = 0 (shown) and the half-lives were determined using a one-phase exponential decay model. Data normalized to DNA mass are shown.

Fig 4. Levels of HIV DNA, 2-LTR circles, and blocks to transcription differ between CD4+ T cells from the gut and blood.

(A) Levels of HIV DNA (Long LTR region) and 2-LTR circles were quantified in CD4+ T cells from the blood and rectum using ddPCR and expressed as copies per million CD4+ T cells (normalized by TERT). (B) The average levels per provirus of each transcript (ratio of each HIV RNA to the Long LTR HIV DNA) were measured in CD4+ T cells isolated from the blood and rectum (n = 7 matched individuals). (C) The ratio of TAR to Long LTR RNA was determined to compare the block to transcriptional elongation in CD4+ T cells from the blood and rectum. (D) The ratio of Read-through to Long LTR RNA was determined to assess the contribution of transcriptional interference to the block to HIV transcription initiation in rectal CD4+ T cells relative to peripheral CD4+ T cells. Bars represent medians. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.

Fig 5. Blocks to HIV transcription differ in the gut and blood.

Multiple blocks to HIV transcription occur in vivo and these differ in CD4+ T cells from the rectum and peripheral blood. Selected examples of potential factors are shown.