Dynamics of HIV latency and reactivation in a primary CD4+ T cell model

Abstract

HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.

Figure 1. Viral production from latently infected cells ex vivo.

Panel A. HIV RNA copies/ml measured in the supernatant of resting CD4+ T cells isolated from treated HIV+ individuals (S: cells from single individuals, P: pooled cells from 2-3 individuals) and stimulated ex vivo with DMSO (control, black), SAHA (blue) or TCR (green) for 1, 2 or 4 days. Panel B. Box plot showing HIV RNA copies/ml measured in the supernatant of resting CD4+ T cells isolated from treated HIV+ individuals and stimulated ex vivo with DMSO (control, black), SAHA (blue) or TCR (green) for 1, 2 or 4 days. Each circle represents a different cellular sample. Bars: min to max. Solid line: median. Panel C. Immunoblot of histones (1 µg) extracted from stimulated ex vivo cells isolated from treated HIV+ individuals. H3K27Ac: anti-histone 3, acetylated Lysine 27; H3K9Ac: anti-histone 3, acetylated Lysine 9; H3: anti-histone 3 (total).

Figure 2. Dynamics of HIV latency in a primary CD4+ T cell model.

Viral-encoded GFP expression (green) and activation marker IL2Rα (CD25) expression (black) during the steps of entry into latency (week -3 to week 2), maintenance of latency (week 4 to 10), and reactivation upon TCR stimulation (8, 24, and 72 hours after week 10). Shown are the geometric mean fluorescence of intensity (geoMFI) ratios of HIV-based vector infected cells and mock non-infected control cells.

Figure 3. Features of HIV transcription.

Panel A. Distribution of HIV reads along the vector genome. On the top is depicted the viral vector genome used (NL4-3-Δ6-drEGFP). Red crosses indicate the genes that are disrupted by stop codon insertion, frameshift or deletion. TSS: transcription start site; D: splice donor; A: splice acceptor. Reads mapping to the LTR are equally assigned to 5′ and 3′ ends, explaining the presence of viral reads upstream the TSS. Panel B. Pattern of splicing for the main viral RNA forms: genomic unspliced full-length viral RNA (US, blue), singly spliced RNAs without the Gag-Pol major intron (SS, green; spliced in D1 but not in D4), and multiply spliced subgenomic mRNAs (MS, red; spliced in D1 and in D4).

Figure 4. Host transcriptional response in latency and reactivation in a primary CD4+ T cell model.

Panel A. Illustrated are 9729 genes with statistically significant association to at least one of the two experimental conditions, HIV infection and TCR stimulation, as evaluated by 2-way analysis of variance of 14513 expressed genes (FDR<0.05). Data are presented in three non-exclusive groups: those regulated in concordance with viral presence (345 genes, top panel), those regulated in concordance with TCR-stimulation 9647 genes, (middle panel), and those showing non-additive synergetic effects by viral presence and TCR stimulation (59 genes, bottom panel). The bold lines represent the group average, and color intensities are proportional to statistical significance of the effects within each group. Panel B. Principal component analysis of host whole-transcriptome data supports a three-step model of entry-latency-reactivation. There is a transcriptionally coherent latency phase between week 4 and week 10, and a distinct reactivation phase at 8, 24, and 72 hours after TCR stimulation. All data, including differentially expressed genes and enrichment analysis, are available at http://litchi.labtelenti.org.

Figure 5. Response to latency reactivating agents.

Panel A. Viral transcription (proportion of HIV transcripts to total cellular transcripts). Panel B. Viral expression (GFP) profiles after reactivation with the various compounds. Panel C. HIV GFP protein expression corrected by the proportion of HIV transcript reads in the cell, normalized by DMSO 8 h. The reference lines represent the W0 of latency, and DMSO 8 hour values.

Figure 6. Reactivating agents fail to provide cellular environment necessary for completion of HIV life cycle.

Each panel summarizes over-represented pathways among the differentially expressed genes induced by the indicated treatment. Organized under ten major categories, each individual circle represents one enriched pathway in Reactome (see methods). The size is proportional to the adjusted p-value (q-value), and the y-axis corresponds to the average effect of the differentially expressed genes within the reported pathway. Only TCR stimulation using anti-CD3/CD28 and IL-2 leads to upregulation of numerous cellular pathways, as well as upregulation of reported HIV cofactors and HIV related pathways (shown in red). AZA is not shown as no enrichment was observed.