High titres of R5-tropic HIV.EGFP reporter virus are needed to infect resting CD4+ T-cells.

<p>Resting CD4+ T-cells from 4 donors were labelled with eFluor670 proliferation dye and cultured without (<i>red</i>) or with autologous mDCs at 1 mDC:10 T-cell ratio (<i>blue</i>) for 24 hours. Cells were incubated with increasing TCID₅₀ per cell of HIV^{NL4.3(AD8)-EGFP} for 2 hours and cultured for 5 days in a low concentration of IL-2 (2 U/ml). 5 days post-infection, cultures were analysed for EGFP expression by flow cytometry to quantify productive infection (A). Non-proliferating eFluor670^hiEGFP- cells were also sorted and cultured for 3 days with anti-CD3/anti-CD28 plus IL-7 to induce EGFP expression from latent infection (B). The percentage of EGFP+ cells per 10⁴ cultured cells is shown. Columns represent the median of donors tested with each donor shown as a different symbol.</p

Latency is established with higher efficiency following co-culture of resting CD4+ T-cells with mDC.

<p>Resting CD4+ T-cells from 2 donors were labelled with eFluor670 cytoplasmic dye and cultured for 24 hours either untreated (<i>red</i>), with 100 nM CCL19 (<i>purple</i>) or with autologous mDCs at 1 mDC:10 T-cell ratio (<i>blue</i>). Cells were incubated with increasing TCID₅₀ per cell of X4-tropic HIV^NL4.3-EGFP. After 2 hours, cells were washed, cultured for 5 days in a low concentration of IL-2 (2 U/ml) and analysed for EGFP expression by flow cytometry to quantify productive infection (A). Additionally, non-proliferating eFluor670^hiEGFP- cells were sorted, cultured for 3 days with anti-CD3/anti-CD28 plus IL-7, in the presence of the integrase inhibitor L-870812 (L8), to induce EGFP expression from post-integration latent infection (B). As a comparative control, aliquots of sorted cells were also cultured for 3 days with L-870812 (L8) but no reactivation stimuli in order to measure background, spontaneous EGFP expression during 3 further days of culture (C). The true level of post-integrated latency in cultures was calculated by subtracting the percentage of EGFP+ cells in the spontaneous cultures from the percentage of EGFP+ cells in reactivated cultures (D). The percentage of EGFP+ cells per 10⁴ cultured cells is shown. Columns represent the median of donor pairs with each donor shown as a different symbol.</p

<i>Ex vivo</i> response of patient-derived HIV LTRs to HDACi in A. SVG and B. HeLa cell lines.

<p>(I) Each cell line was incubated with different concentrations of HDACi for 24 hr and toxicity was measured by the MTS assay. The cytotoxic concentration 50 (CC₅₀) for each drug is shown. (II) SVG or HeLa cells were transiently transfected with the wild-type NL4-3 LTR- pCEP4 and treated with various concentrations of HDACi for 24 hr. Co-transfection with Tat (4 ng) or incubation with PMA (20 nM) were used as positive controls. The activity of the LTR was measured as the fold change in luciferase compared to the untreated sample. # indicates doses of individual HDACi that were closest to the CC₅₀ and induced the largest fold change increase in luciferase activity. (III) Luciferase expression following transfection of pCEP4 containing LTR sequences isolated from total memory CD4⁺ T-cells prior to or after cART and treated with the optimal dose of HDACi. (C) Comparison of the luciferase expression in HeLa (red) and SVG (blue) following transfection of pCEP4 containing LTR sequences from (B III) and treated with the optimal dose of HDACi. Boxes represent the median, 25th and 75th percentiles and error bars the 10th and 90th percentiles. Ns  =  not statistically significant.</p

Phylogenetic analyses of DNA sequences derived from integrated virus in CD4⁺ memory T-cells.

<p>Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4⁺ T-cells prior to the initiation of cART (<i>open symbols</i>), after at least 18 months of cART (<i>closed symbols</i>) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of &gt;75 are shown on branches. All hypermutated clones (<i>P</i>&lt;0.05 analysed on Hypermut V2.0) were excluded from the analysis.</p

Primers and triple-nested Alu-LTR-PCR conditions.

<p>Primers and triple-nested Alu-LTR-PCR conditions.</p

Patient characteristics.

<p>BL, Baseline; VL, viral load in plasma; * age at recruitment ie BL.</p><p>Patient characteristics.</p

Vorinostat synergises with Tat and not PMA to increase transcription of the HIV LTR.

<p>SVG cells were transiently transfected with pCEP4 plasmid containing either NL4-3 or patient-derived HIV LTRs and luciferase activity was quantified following treatment with vorinostat (5 µM) and co-transfection with Tat using (A) non-mutated or (B) hypermutated (<i>P</i>&lt;0.05 analysed on Hypermut V2.0) patient-derived HIV LTRs. (C) luciferase activity was quantified following stimulation of non-mutated patient-derived LTRs with vorinostat and PMA (20 ng/ml). Error bars represent standard error of the mean of three independent experiments. * <i>P</i>&lt;0.05, ** <i>P</i>&lt;0.01 and *** <i>P</i>&lt;0.001. ns  =  not statistically significant.</p

Cloning of patient-derived HIV LTRs into pCEP4.

<p>(A) Total memory T cells from HIV-infected individuals were isolated from blood collected prior to or after receiving cART. The integrated HIV LTRs from these cells were isolated by triple nested Alu-LTR PCR. (B)(I) The plasmid pCEP4 was digested with <i>Sal</i>I to remove the entire PCMV promoter region and the SV40 poly A sequences. (II) DNA sequence of the Δ-57/-4 HIV LTR and the luciferase gene was generated by digestion of the Δ-57/-4 LTR pGL3-Basic vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113341#pone.0113341-Gray1" target="_blank">[20]</a> and ligated into pCEP4. (III) Patient-derived HIV LTRs were cloned into the Δ-57/-4 LTR-pCEP4 vector using the <i>Acc</i>6<i>5</i>I and <i>Hind</i>III sites. (IV) Patient LTR pCEP4 was transfected into SVG and HeLa cell lines; the activity of various HDACi on LTR transcription was measured by quantification of luciferase activity.</p