CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

Abstract

Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a "shock and kill" strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs.

Figure 1. Screening of single guide RNAs (sgRNAs) that guide dCas9-transcriptional activator to the HIV-1 long terminal repeat (LTR) promoter.

(a) Illustration of sgRNA-guided HIV-1 genome regulation platform for dCas9 fusion with transcriptional activator (VP64). (b) Diagram of dCas9-VP64 expressing lentiviral or retroviral vector and sgRNA expressing lentiviral vector (LV). (c) Illustration of sgRNA targeting locations within HIV-1 LTR. Some of transcription factors are shown in different colors. (d,e) Identification of effective sgRNAs with dCas9-VP64 in HEK293T cells transiently coexpressing NL4-3-EcoHIV-firefly luciferase (eLuc) reporter (d) or TZM-bI cells integrated with LTR-eLuc reporter (e). At 2 days after cotransfection, ONE-Glo™ luciferase assay was performed. Data represent mean ± SEM of 4 independent transfections, showing fold changes in luminescent reporter activity relative to corresponding empty sgRNA expression vector (LTR-0). *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Figure 2. Screening of MS2-mediated sgRNAs (msgRNAs) targeting the HIV-1 long terminal repeat (LTR) in the dCas9-synergistic activation mediator (SAM) system.

(a) Illustration of SAM-VPH complex derived from dCas9-VP64 (V) and MS2-p65-HSF1 (PH) with msgRNA. (b) Similar activation of transfected EcoHIV-eLuc reporter by inducible (TRE) or constitutive (LTR) dCas9-VP64 expression in HEK293T cells. (c) Similar reactivation of latent HIV-LTR-eLuc reporter in TZM-bI cells after either transient plasmid transfection by Lipofectamine 3000 or stable infection by lentivirus. Note that MS2-p65-HSF1 alone did not influence the basal activity of LTR-eLuc reporter. The msgRNA-J has no effect due to 2 nucleotide mismatches to the LTR sequence from TZM-bI cells. (d) Identification of msgRNA LTR-L and O with best efficiency to activate EcoHIV-eLuc reporter in HEK239T cells with dCas9-SAM system. Data represent mean ± SEM of 4–6 independent transfections or infections, showing fold changes relative to corresponding empty msgRNA expression vector (LTR-0). *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Figure 3. Robust reactivation of HIV-1 LTR promoter by lentivirus-mediated dCas9-SAM in HIV-1 latent cell lines.

(a,b) TZM-bI cells were infected with dCas9-VPH and indicated msgRNA lentiviruses at 1 or 10 MOI and luciferase activity was measured at 2–21 days post-infection (dpi). (c,d) Indicated HIV-1 EGFP reporter T cell lines were infected with dCas9-VPH and indicated msgRNAs lentiviruses at 10 MOI and flow cytometric analysis for EGFP percentage (c) and individual intensity (d) was performed at 1 dpi. (e,f) HIV-1 latent CHME5 microglial cells were infected at 10 MOI and flow cytometry was performed at 1 dpi. All data represent relative changes (n = 3–4) to the corresponding empty msgRNA control (Zero). *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Figure 4. Potent and persistent reactivation of HIV-1 LTR promoter induces suicide death of HIV-1 latent 2D10 T and CHME5 microglial cells but not TZM-bI cells due to the production of toxic viral proteins.

(a,b) TZM-bI cells were infected with indicated lentiviruses for 1 d and triple antibiotic selection with puromycin/hygromycin/zeoin (P/H/Z) was maintained for 4 d before passage, OneGlo luciferase reporter assay (a) and CellTiter-Glo® luminescent cell viability assay (b). (c,d) CHME5 cells were infected with indicated lentiviruses and cultured for 5 d in the absence or presence of P/H/Z triple selection. Flow cytometry analysis was performed for reactivation efficiency (c) and total survival cell number (d). (e) Caspase-Glo^® 3/7 Assay was performed at 2 dpi to evaluate apoptotic cell death. The reactivation efficiency was validated by flow cytometry (EGFP % on the bottom). (f,g) The 2D10 cells were infected for 1 and 4 d, and the reactivation efficiency (f) and relative total survival cell number (g) were determined by flow cytometry. *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Figure 5

Potent and persistent reactivation of HIV-1 EGFP reporter virus and suicide cell death by dCas9-VPH/msgRNA in CHME5 microglial cells (a) and Jurkat-derived 2D10 (b) or E4 (c) T cell lines. (a) Time-dependent reduction in EGFP⁺ CHME5 cells due to continuous reactivation and suicide cell death. P/H/Z triple selection enriched lentivirus-infected cells and validated continuous EGFP reporter reactivation. **p < 0.01 indicate statistical significance by student’s t test as compared to corresponding non-selection groups. (b,c) Time-dependent reduction in EGFP⁺ 2D10 or E4 cells due to continuous reactivation and suicide cell death.

Figure 6. Robust reactivation of HIV-1 LTR by dCas9-SAM/msgRNAs but not SAHA in TZM-bI and CHME5 cells.

(a–c) The dCas9-VPH stable TZM-bI cells were treated with SAHA or infected with indicated msgRNA lentivirus for 2–4 d before luminescence reporter assay (a) and CellTiter-Glo^® luminescent cell viability assay (b). Relative reactivation change was normalized by cell viability (c). (d–g) The dCas9-VPH stable CHME5 cells were treated with SAHA or infected with indicated msgRNA lentivirus for 4 d before EGFP flow cytometry for reactivation efficiency (d), total (e) and individual (f) fluorescent intensity and survival cell number (g). All data represent mean ± SEM of 3–4 independent experiments. *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test as compared with corresponding vehicle control or LTR-zero control.

Figure 7. Overview of the dCas9-VPH SAM system using MS2-mediated sgRNA to direct multiple activators to the enhancer region of the HIV-1 promoter and reactivate viral protein expression in HIV-1 latent cells, which further induce suicide death and/or trigger host immune response to kill HIV-1 latent cells.