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. 2013 Apr;87(8):4716-27.
doi: 10.1128/JVI.03478-12. Epub 2013 Feb 13.

A doubly fluorescent HIV-1 reporter shows that the majority of integrated HIV-1 is latent shortly after infection

Matthew S Dahabieh  1 , Marcel Ooms, Viviana Simon, Ivan Sadowski
Affiliations

A doubly fluorescent HIV-1 reporter shows that the majority of integrated HIV-1 is latent shortly after infection

Matthew S Dahabieh et al. J Virol. 2013 Apr.

Abstract

HIV-1 latency poses a major barrier to viral eradication. Canonically, latency is thought to arise from progressive epigenetic silencing of active infections. However, little is known about when and how long terminal repeat (LTR)-silent infections arise since the majority of the current latency models cannot differentiate between initial (LTR-silent) and secondary (progressive silencing) latency. In this study, we constructed and characterized a novel, double-labeled HIV-1 vector (Red-Green-HIV-1 [RGH]) that allows for detection of infected cells independently of LTR activity. Infection of Jurkat T cells and other cell lines with RGH suggests that the majority of integrated proviruses were LTR-silent early postinfection. Furthermore, the LTR-silent infections were transcriptionally competent, as the proviruses could be reactivated by a variety of T cell signaling agonists. Moreover, we used the double-labeled vector system to compare LTRs from seven different subtypes with respect to LTR silencing and reactivation. These experiments indicated that subtype D and F LTRs were more sensitive to silencing, whereas the subtype AE LTR was largely insensitive. Lastly, infection of activated human primary CD4(+) T cells yielded LTR-silent as well as productive infections. Taken together, our data, generated using the newly developed RGH vector as a sensitive tool to analyze HIV-1 latency on a single-cell level, show that the majority of HIV-1 infections are latent early postinfection.

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Figures

Fig 1
Fig 1
Schematic representation of the Red-Green-HIV-1 (RGH) vector. (A) HIV-1 B-LAI Δenv is labeled with eGFP as an in-frame gag fusion flanked by HIV-1 protease cleavage sites (inverted triangles), and by a CMVIE-driven mCherry cassette located in place of nef. WT, wild type. (B) Schematic depiction of RGH infection of target cells and the resultant fluorescent protein profiles over time. (C) Schematic depiction of HIV-1 RGH-infected cell populations detected by FACS analysis.
Fig 2
Fig 2
LTR silencing in RGH-infected Jurkat cells occurs early and requires integration. (A) Flow cytometry time course of RGH-infected Jurkat cells. Plots shown are representative of the results of triplicate infections. p.i., postinfection. (B) Plot of positive cells from each colored fraction (eGFP+ mCherry−, eGFP− mCherry+, eGFP+ mCherry+) over the course of infection. Error bars represent standard deviations of the results of triplicate experiments. (C) Data from the experiment represented by panel B are enumerated as ratios of red cells to yellow cells (proportion of eGFP− mCherry+ to eGFP+ mCherry+). Error bars represent standard deviations of the results of triplicate experiments. ns, nonsignificant; *, P < 0.05 (Student's t test). (D) Jurkat cells were infected with RGH in the presence of the integrase inhibitor raltegravir (10 μM). Cells were analyzed by flow cytometry over a period of 7 days. Error bars represent standard deviations of the results of triplicate experiments. (E) Jurkat cells were infected as described for panel D, except that an RGH variant bearing a catalytically defective integrase variant (D116A) was used. Additionally, raltegravir was excluded.
Fig 3
Fig 3
Identification of LTR-silent RGH infections is dependent on both the LTR and CMV promoters, and is not specific for Jurkat T cells. (A) Schematic representation of RGH variant viruses. Gag-eGFP lacks the CMV-mCherry construct, while ΔU3 and ΔCMV both contain both fluorescent coding sequences (eGFP and mCherry) but lack U3 in the 3′ LTR and the CMV promoter, respectively. Variants are otherwise isogenic. (B) Jurkat cells were infected with WT and variant RGH and analyzed by flow cytometry 4 days postinfection. Plots shown are representative of the results of triplicate experiments. (C) Fluorescence microscopy (×100) of RGH-infected Jurkat cells (4 days postinfection). eGFP− mCherry+ and eGFP+ mCherry+ cells are indicated in the merge panel by red and yellow arrows, respectively. Images shown are representative of the results of triplicate experiments. (D) SupT1, U937, HEK293T, and HeLa cells were infected with RGH and analyzed by flow cytometry 4 days postinfection. Plots are representative of the results of duplicate experiments.
Fig 4
Fig 4
Silent LTRs in RGH-infected cells are transcriptionally competent. (A) RGH-infected Jurkat cells (4 days postinfection) were treated with DMSO, TNF-α, PMA, PMA/Iono, 5-aza-dC, SAHA, TSA, or TNF-α/SAHA for 24 h prior to analysis by flow cytometry. Plots shown are representative of the results of triplicate experiments. (B) Data from panel A are enumerated as the red/yellow ratio of the infected population. Error bars represent standard deviations of the results of triplicate experiments. ns, nonsignificant; **, P < 0.01; ***, P < 0.001 (Student's t test). (C) RGH-infected Jurkat cells were sorted to greater than 90% purity 3 days postinfection. The left panel depicts the Jurkat cells infected with RGH at day 3 postinfection, and the right panel shows the purity of the individual cell populations after sorting (composite of three individual FACS plots). Cells were left to recover for 24 h prior to treatment with either DMSO or TNF-α for a further 24 h, followed by analysis by flow cytometry. (D) Data from the experimental outline in panel C. Cells were analyzed by flow cytometry. Error bars represent standard deviations of the results of duplicate experiments. inf., infected. **, P < 0.01; ***, P < 0.001 (Student's t test).
Fig 5
Fig 5
RGH recapitulates differences in latency observed between HIV-1 group M subtypes. (A) Schematic representation of RGH variants containing promoter sequences from the major group M viral subtypes. Viruses are isogenic except for a 209-bp BseAI-AflII fragment containing the core promoter region of the LTR that stretches from position −147 to position +68. (B) Jurkat cells were infected with the subtype RGH variants and analyzed by flow cytometry 4 days postinfection. Plots shown are representative of the results of triplicate experiments. (C) Jurkat cells were infected with the subtype RGH variants. At 4 days postinfection, cells were treated with either DMSO or PMA/ionomycin for 24 h and then analyzed by flow cytometry. Error bars represent standard deviations of the results of triplicate experiments. (D) Data from panel C are enumerated as fold change between the PMA and DMSO treatments. Error bars represent standard deviations of the results of triplicate experiments. R, red; Y, yellow; ns, nonsignificant; ***, P < 0.001 (Student's t test).
Fig 6
Fig 6
LTR-silent RGH infections occur in primary CD4+ T cells. (A) Activated (IL-2 + PHA) primary CD4+ T cells from three healthy donors were infected with purified RGH. Infected cells were cultured in IL-2-containing media for 6 days prior to FACS analysis. (B) Nonactivated (IL-2) primary CD4+ T cells from three healthy donors were infected with 10-fold more virus than in the experiment represented by panel A. Infected cells were cultured in IL-2-containing media for 6 days prior to FACS analysis. (C) Data from panel A are displayed graphically, with the ratio of red cells to yellow cells indicated at the bottom.
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