Analysis of human immunodeficiency virus type 1 transcriptional elongation in resting CD4+ T cells in vivo

Abstract

A stable latent reservoir for human immunodeficiency virus type 1 (HIV-1) in resting memory CD4+ T cells presents a barrier to eradication of the infection even in patients on highly active antiretroviral therapy. Potential mechanisms for latency include inaccessibility of the integrated viral genome, absence of key host transcription factors, premature termination of HIV-1 RNAs, and abnormal splicing patterns. To differentiate among these mechanisms, we isolated extremely pure populations of resting CD4+ T cells from patients on highly active antiretroviral therapy. These cells did not produce virus but retained the capacity to do so if appropriately stimulated. Products of HIV-1 transcription were examined in purified resting CD4+ T cells. Although short, prematurely terminated HIV-1 transcripts have been suggested as a marker for latently infected cells, the production of short transcripts had not been previously demonstrated in purified populations of resting CD4+ T cells. By separating RNA into polyadenylated and nonpolyadenylated fractions, we showed that resting CD4+ T cells from patients on highly active antiretroviral therapy produce abortive transcripts that lack a poly(A) tail and that terminate prior to nucleotide 181. Short transcripts dominated the pool of total HIV-1 transcripts in resting CD4+ T cells. Processive, polyadenylated HIV-1 mRNAs were also present at a low level. Both unspliced and multiply spliced forms were found. Taken together, these results show that the nonproductive nature of the infection in resting CD4+ T cells from patients on highly active antiretroviral therapy is not due to absolute blocks at the level of either transcriptional initiation or elongation but rather relative inefficiencies at multiple steps.

FIG. 1.

Purification and characterization of resting CD4⁺ T cells from aviremic patients on highly active antiretroviral therapy. (A) Flow cytometric analysis of unfractionated PBMCs (left panel) and highly purified resting CD4⁺, DR⁻ T cells (right panel) stained with phycoerythrin-conjugated anti-CD4 and fluorescein isothiocyanate-conjugated anti-HLA-DR antibodies. (B) Production of virus particles by purified resting CD4⁺ T cells. Resting CD4⁺ T cells were cultured in medium alone (open diamonds) or with anti-CD3 and anti-CD28 monoclonal antibodies (solid diamonds). Virus particle concentrations in the supernatant were measured by RT-PCR at the indicated times.

FIG. 2.

Detection of nonpolyadenylated and polyadenylated HIV-1 RNAs associated with resting CD4⁺ T cells from patients on highly active antiretroviral therapy. (A) Schematic organization of the HIV-1 long terminal repeat with location of PCR primers. The HIV-START.2 and HIV-SHORT3′ primer set amplifies both abortive and full-length products of transcription. The HIV-START.2 and HIV-LONG3′ primer set amplifies any full-length product of transcription. (B) Southern blots of RT-PCR products amplified from nonpolyadenylated HIV-1 RNA isolated from 10⁶ resting CD4⁺ T cells with HIV-START.2 and either the HIV-SHORT3′ or HIV-LONG3′ primer. Patient samples were analyzed in duplicate with (+) and without (−) reverse transcriptase (RT). (C) Southern blots of RT-PCR products amplified from polyadenylated HIV-1 RNA isolated from 10⁶ resting CD4⁺ T cells with HIV-START.2 and either the HIV-SHORT3′ or HIV-LONG3′ primer. Patient samples were analyzed in duplicate with (+) and without (−) reverse transcriptase. (D) Southern blots of RT-PCR products amplified from nonpolyadenylated (top) and polyadenylated (bottom) HIV-1 RNA isolated from 10⁶ PBMCs isolated from a representative viremic patient. Amplification was carried out with the HIV-SHORT3′ primer pair.

FIG. 3.

Analysis of nonpolyadenylated and polyadenylated HIV-1 RNAs at various cell dilutions. (A) Nonpolyadenylated RNA (top panels) and polyadenylated RNA (bottom panels) were isolated from aliquots of 10⁶, 0.5 × 10⁶, 0.25 × 10⁶, and 10⁵ resting CD4⁺ T cells. Southern blots of RT-PCR products at each cell dilution with HIV-START.2 and HIV-SHORT3′ primers are shown. (B) Southern blots of RT-PCR products amplified from HIV-1 RNA isolated from 10⁶, 0.5 × 10⁶, 0.25 × 10⁶, and 10⁵ PBMCs from viremic patients.

FIG. 4.

Schematic of the HIV-1 genome and locations of nested HIV-1 primers. The primers used to prime cDNA synthesis are shown as E5RT1 and E7RT3. Four different primer sets were used: US is able to amplify unspliced HIV-1 only, and E2 is able to amplify unspliced HIV-1 RNA or spliced HIV-1 RNAs. With E2, HIV-1 RNAs that are unspliced or contain exon 2A, 2AE, 3A, or 3A will produce a larger band, while HIV-1 RNAs that contain exon 2 or 3 and 4, 4A, 4B, 4C, or 5 will produce a smaller band. The E5 and FL primer sets can only amplify multiply spliced HIV-1 RNAs. cDNA synthesized with E5RT1 was amplified with the E2 and US sets. cDNA synthesized with E7RT3 was amplified with E5 and FL.

FIG. 5.

HIV-1 RNA exon usage in transcripts detected from resting CD4⁺ T cells isolated from patients on highly active antiretroviral therapy.