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. 2013 Nov;446(1-2):283-92.
doi: 10.1016/j.virol.2013.07.037. Epub 2013 Sep 6.

Dual-color HIV Reporters Trace a Population of Latently Infected Cells and Enable Their Purification

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Dual-color HIV Reporters Trace a Population of Latently Infected Cells and Enable Their Purification

Vincenzo Calvanese et al. Virology. .
Free PMC article

Abstract

HIV latency constitutes the main barrier for clearing HIV infection from patients. Our inability to recognize and isolate latently infected cells hinders the study of latent HIV. We engineered two HIV-based viral reporters expressing different fluorescent markers: one HIV promoter-dependent marker for productive HIV infection, and a second marker under a constitutive promoter independent of HIV promoter activity. Infection of cells with these viruses allows the identification and separation of latently infected cells from uninfected and productively infected cells. These reporters are sufficiently sensitive and robust for high-throughput screening to identify drugs that reactivate latent HIV. These reporters can be used in primary CD4 T lymphocytes and reveal a rare population of latently infected cells responsive to physiological stimuli. In summary, our HIV-1 reporters enable visualization and purification of latent-cell populations and open up new perspectives for studies of latent HIV infection.

Figures

Figure 1
Figure 1. Two-Color Viruses Identify a Population of Latently Infected Cells
(a) Diagram of the derivation of two-color viruses from the original strains, with a scheme of a classical single reporter virus bearing the reporter gene in the nef ORF. (b) In the 89mASG construct, EGFP is under the control of the SFFV promoter and mApple was added upstream of the transcriptional unit in the position of the nef ATG (see d). (c) In the R7GEmC construct, GFP replaces nef and a whole transcriptional unit (EF1α-mCherry) was inserted downstream. (d) Alignment of regions of interest (from the end of env to the 3′LTR) of the original 89.6 and HXB2 strains deposited in the NCBI database (U39362.2 and NC001802), the starting plasmid and final constructs for each of the HIV clones 89mASG (d, top) and R7GEmC (d, bottom). Insertion points of the first reporter in the nef ORF, the promoter (SFFV or EF1α, respectively) and the second reporter, followed by the end of nef and 5′LTR. (e,f) Cytometric analysis of Jurkat T cells 3 days post-infection, with the indicated titers of 89mASG (e) and R7GEmC (f). (g,h) FACS analysis of 89mASG (g, left) and R7GEmC (h, left) Jurkat T cells sorted 5 days post-infection. Double-negative (uninfected), double-positive (actively infected) and single-positive (latently infected, expressing GFP- or mCherry-only) cells are shown for both viral strains. Fluorescence microscopy images of 89mASG- (g, right) and R7GEmC- (h, right) infected Jurkat T cells. (i) qPCR quantification of proviral DNA in the sorted Jurkat populations, using the primer-probe combination #E for the HIV-1 gag gene and RNAseP for normalization. (j) qPCR quantification of viral transcripts of unspliced (US) gag, singly- (SS) env and multiply-spliced (MS) tat/rev common regions (#E, F, and G; Suppl. table 2), normalized for cell GAPDH. (k) Western blot quantification of the HIV proteins vif and gag and the endogenous protein α-actin in the sorted Jurkat populations.
Figure 2
Figure 2. Latently Infected Cells Respond to Reactivating Drugs
(a–d) Five days after sorting, expanded single-positive (latent) and double-negative (uninfected) 89mASG (a, c) and R7GEmC (b, d) Jurkat T cells were treated with 10 ng/ml TNFα, 5 μM prostratin or 2.5 μM SAHA, alone or in combination, as indicated. FACS analysis of a representative treatment (a, b) and histogram quantification of percent population in the active gate for three different experiments (c, d). P<0.001, compared to DMSO control for all treatments. P<0.01 for combination vs. single treatments, except for R7GEmC prostratin vs. prostratin+SAHA P<0.05. (e,f) FACS analysis of SupT1 and A301 cell lines, infected with 20 ng p24 of 89mASG (e) or R7GEmC (f) virus. (g) A larger panel of latent HIV-reactivating drugs was used at indicated concentrations on sorted, single positive Jurkat, SupT1 and A301 cells. Histograms show quantification of the percent population in the active gate. Data indicate mean ± SD for three different treatments. P<0.01, compared to DMSO 0.1% control for all treatments, except for: SAHA 0.1 not significant (n.s.) in all combinations; SAHA 0.1 n.s. in SupT1 and A301-R7GEmC, P<0.05 in Jurkat-R7GEmC; Bryostatin 30 P<0.05 in A301-R7GEmC; HMBA 0.3 n.s. in all combinations; HMBA 1 n.s. in A301 and SupT-R7GEmC, P<0.05 in Jurkat-R7GEmC and SupT-89mASG; PHA-M n.s in A301-89mASG and P<0.05 in A301-R7GEmC.
Figure 3
Figure 3. High-Throughput Screening Identifies New Reactivator Drugs Using Two-Color HIV Constructs
Sorted 89mASG-infected, GFP-only-positive Jurkat cells were used to test the Tocriscreen drug library for latent HIV reactivation (see Methods). (a) Dot plot showing quantification of total blue area (Hoechst 33342 signal, total cell) against total red area (mApple signal, reactivated HIV). Red dots indicate the position of untreated wells; the shaded area indicates the median ± 2SD of the red/blue area ratio for negative control wells. Green dots indicate positive control wells, treated with 10 ng/ml TNFα, 5 μM prostratin or 2.5 μM SAHA and their combinations. Blue circle shows novel reactivators identified in the screening (see Suppl. Table 1). (b) Validation of representative hits by FACS analysis. Apomorphine and its analog R-(−)-propylnorapomorphine (PNA), ritanserin, clozapin, AG555, AG18, piceatannol, pterostilbene, dilazep, dipyridamole, terreic acid (TA), simvastatin and NSC95397 were used to validate hits and pathways identified by drug screening. Representative FACS plots of sorted single-positive Jurkat T cells for both viruses. The productive gate contains the reactivated cells. In the last plot on the right, NSC95397 treatment shows alteration of the fluorescent profile by the drug fluorescence. (c,d) Histogram plot of percent population in the productive gate (grey) for each treatment and percentage of cells in the live gate (light blue) for sorted single-positive Jurkat T cells for 89mASG (c) and R7GEmC (d) reporters. Data shown as mean ± SD for three different treatments. For each compound, a grey triangle between the histograms indicates decreasing drug concentration (30, 10 and 3 μM). Treatments for which cell death was predominant are labeled TOXIC. Ritanserin 30μM, clozapin 30μM, AG555 30, 10 and 3μM, piceatannol 30 and 10μM, pterostilbene 30μM, dilazep 30μM, TA 10 and 3μM (P<0.01) and PNA 30μM, piceatannol 3μM, pterostilbene 10μM, dipyridamole 30μM (P<0.05) show significant reactivation of the latent 89mASG virus compared to the DMSO control. Apomorphin 30μM, PNA 30μM, ritanserin 30μM, clozapin 30μM, AG555 30, 10 and 3μM, AG18 30, 10 and 3μM, piceatannol 30 and 10μM, pterostilbene 30 10 and 3 μM, TA 10μM (P<0.01) and PNA 10μM, ritanserin 10μM, piceatannol 3μM, dilazep 30, 10 and 3μM, TA 3μM (P<0.05) show significant reactivation of the latent R7GEmC virus compared to the DMSO control. (e) FACS analysis of drug treatments combined with 0.5 μM SAHA, 0.5 μM prostratin and 0.5 ng/ml TNFα. Representative FACS plots of sorted GFP-only-positive Jurkat T cells for 89mASG treated with DMSO (control), SAHA (0.5 μM) alone or with terreic acid (10 μM) and dilazep (30 μM). (f) Histogram plot of percent population of the sorted cells in the productive gate for each treatment at indicated concentrations (μM). Dilazep 30, 10μM and piceatannol 10μM show significant (P<0.01) increase in reactivation 0.5 μM SAHA, 0.5 μM prostratin and 0.5 ng/ml TNFα as compared to the reactivation obtained with these three drugs alone. TA also shows positive interaction with SAHA and prostratin at 10μM (P<0.01) and with SAHA at 3μM(P<0.05). AG555 interact enhances reactivation mediated by prostratin and TNFα (P<0.01), whilst dipyridamole 30 and 10μM shows significant (P<0.05) positive interaction with prostratin only.
Figure 4
Figure 4. Two-Color Viruses Label a Population of Latently Infected Primary T Cells
(a) FACS analysis of activated CD4+ T cells from two human donors (#1, #2) infected with R7GEmC at indicated virus amounts. (b) Fluorescence microscopy image of infected CD4+ T cells showing mCherry-positive (latent, red) and GFP-positive (active, green) cells. (c) qPCR quantification of proviral DNA in the sorted populations, using primer-probe combinations for the HIV-1 gag gene and the RNAseP gene for normalization. Data represent mean ± SEM for three different donors. (d) Western blot quantification of HIV gag and cell α-actin in sorted CD4+ T cell populations from pooled donors. (e) FACS analysis of sorted CD4+ T cell populations from donors #1 and #2 after 48 h with anti-CD3/CD28-coated Dynabeads (αCD3/28) or left untreated (CTR). Right, histogram plot for the mCherry-only population of each donor in which the treated profile (green line) is overlaid on the untreated (grey shaded). (f) Dot plot of mCherry-only population from three pooled donors treated with 10 ng/m, TNFα, 1 μM prostratin, 1 μM SAHA or anti-CD3/CD28-coated Dynabeads (1 bead/cell).

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