Journal: The Journal of Clinical Investigation

Article Title: HIV latency is reversed by ACSS2-driven histone crotonylation

doi: 10.1172/JCI98071

Figure Lengend Snippet: (A) The J-Lat A1 cells were treated overnight with 30 mM Na-Cro (n = 3), 6 nM PEP005 (n = 3), 2 μM JQ1 (n = 4), 10 nM bryostatin (n = 4), 250 nM vorinostat (n = 3), 2 μM GSK343 (n = 4), 5 μM AZA-dC (n = 4), or 5 ng/ml PMA (n = 4) individually or in combination as indicated in the panel and evaluated for HIV reactivation by RT-qPCR. *P < 0.05 and ***P < 0.001 versus LRA alone. (B) The J-Lat A1 cells were pretreated with 250 nM of vorinostat; then 40 mM of Na-Cro was added overnight. The HIV reactivation was measured by real-time PCR. **P < 0.01 and ***P < 0.001 versus control treatment (n = 3). NS, not significant compared with Na-Cro treatment alone. (C) Effect of 30 mM Na-Cro, individually or in combination with PEP005, on reactivation of latent HIV was tested. The cells were treated overnight, and J-Lat A1 cells with HIV reactivation were measured by detection of GFP-expressing cells using flow cytometry. ***P < 0.001 versus control treatment; ###P < 0.001 versus PEP005 alone (n = 3). (D) U1 cells were treated similarly as in C; RT-qPCR was performed to measure viral expression. ***P < 0.001 vs. untreated control; ###P < 0.001 versus PEP005 treatment alone (n = 3). The data were analyzed with 1-way ANOVA.

Article Snippet: For reactivation of latent HIV, cells were treated with sodium crotonate (Na-Cro; Sigma-Aldrich), PEP005 (Tocris Bioscience), vorinostat (Santa Cruz Biotechnology), GSK343 (Sigma-Aldrich), or AZA-dC (Santa Cruz Biotechnology) for 4, 18, or 24 hours.

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Control, Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: HIV latency is reversed by ACSS2-driven histone crotonylation

doi: 10.1172/JCI98071

Figure Lengend Snippet: (A–C) Primary CD4+ T cells isolated from HIV-positive patients (n = 6) under ART were treated with 100 ng/ml PMA plus 2 μM ionomycin, 12 nM PEP005, 40 mM Na-Cro, or 12 nM PEP005 combined with 40 mM Na-Cro for 6 hours. Viral transcription from total RNA was analyzed by reverse transcriptase digital droplet PCR with primers targeting initiation (TAR region) (A), elongation (long LTR) (B), or full transcription [poly(A) region] (C) of the HIV genome. (D) Na-Cro synergistically reactivates the latent HIV with PEP005 in primary CD4+ T cells isolated from patients under suppressive ART. The difference between the observed fraction and predicted fraction or faxyo-faxyp (Δfaxy) was determined by Bliss independence analysis from 6 patient samples as described in the Supplemental Methods. **P < 0.01 and ***P < 0.001 versus faxyP (n = 6).

Article Snippet: For reactivation of latent HIV, cells were treated with sodium crotonate (Na-Cro; Sigma-Aldrich), PEP005 (Tocris Bioscience), vorinostat (Santa Cruz Biotechnology), GSK343 (Sigma-Aldrich), or AZA-dC (Santa Cruz Biotechnology) for 4, 18, or 24 hours.

Techniques: Isolation, Reverse Transcription

Journal: Cell Reports Methods

Article Title: A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro

doi: 10.1016/j.crmeth.2021.100122

Figure Lengend Snippet: Effect of drug treatments on viral persistence in the in vitro infection model (A) Assay overview. Schematic representation of the assay protocol involving the HIV-1 NL4-3 -infected cell culture model (Jurkat/NL cells). (B) Changes in supernatant p24 levels without drugs, with 5 nM PEP005 or 50 nM EFdA, or with a combination of 50 nM EFdA and 5 nM PEP005 (n = 11, 9, 11, and 11, respectively). Drug treatment was terminated on week 9, but analysis continued for an additional 8 weeks. (C) Log rank test comparison of the percentage of non-recurrence in the EFdA single treatment and the combination treatment. (D) Changes in supernatant p24 levels in a representative experiment (experiment 1) from experiments shown in Figure 3 B. (E and F) Assessment of the viral rebound in Jurkat/NL cells after drug discontinuation. Cells treated with drugs or untreated cells were stimulated with TNF-α (10 ng/mL) in week 17, and supernatant p24 (E) and intracellular p24 levels (F) were analyzed on day 6 after stimulation. Asterisk (∗) denotes below detection limit.

Article Snippet: PEP005 (PKC activator) was purchased from Cayman Chemical (Ann Arbor, MI); SAHA (vorinostat; HDAC inhibitor) from Santa Cruz Biotechnology (Dallas, TX); JQ-1 (BRD4 inhibitor) from BioVision (Milpitas, CA); GSK525762A (BRD4 inhibitor) from ChemScene (Monmouth Junction, NJ); Ro5-3335 from Merck (Darmstadt, Germany).

Techniques: In Vitro, Infection, Cell Culture, Comparison

Journal: Cell Reports Methods

Article Title: A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro

doi: 10.1016/j.crmeth.2021.100122

Figure Lengend Snippet: Effect of various combinations of antiretroviral drugs and LRAs on viral persistence (A–C) Changes in HIV-1 production under treatment with 5 nM PEP005, 50 nM EFdA, and/or 500 nM DRV (protease inhibitor) (A); 5 nM PEP005 and/or 50 nM DTG (integrase inhibitor) (B); 100 nM EFdA, 500 nM SAHA (HDAC inhibitor), and/or 500 nM prostratin (PKC activator) (C). (D) EFdA at different concentrations (50 nM, 200 nM, or 1 μM) was examined. A higher concentration of EFdA (200 nM and 1 μM) slightly delayed the recurrence of supernatant viruses post treatment interruption. (E–H) Effect of different drug treatment protocols in the WIPE assay. (E) Changes in supernatant p24 levels with 50 nM EFdA for 9 weeks and then FFdA + PEP005 (5 nM) for an additional 9 weeks (n = 6). (F) Analysis of HIV-1 mRNA transcripts after TNF-α stimulation in cells without viral rebound on week 26 (three samples). (G) Changes in supernatant p24 levels with EFdA + PEP005 followed by the EFdA treatment for 9 weeks. (H) Analysis of HIV-1 mRNA transcripts after TNF-α stimulation in cells without viral rebound on week 26 (four samples). Asterisk (∗) represents below detection limit.

Article Snippet: PEP005 (PKC activator) was purchased from Cayman Chemical (Ann Arbor, MI); SAHA (vorinostat; HDAC inhibitor) from Santa Cruz Biotechnology (Dallas, TX); JQ-1 (BRD4 inhibitor) from BioVision (Milpitas, CA); GSK525762A (BRD4 inhibitor) from ChemScene (Monmouth Junction, NJ); Ro5-3335 from Merck (Darmstadt, Germany).

Techniques: Protease Inhibitor, Concentration Assay

Journal: Cell Reports Methods

Article Title: A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro

doi: 10.1016/j.crmeth.2021.100122

Figure Lengend Snippet: HIV-1 infection dynamics with antiviral drugs in the WIPE assay (A) Fitting of the mathematical model to the experimental data in the WIPE assay without and with antiviral drug(s): numbers of uninfected and latently infected cells (cells/mL), virus-producing cells (cells/mL), supernatant p24 (pg/mL), and normalized proviral DNA. The shadowed regions correspond to 95% posterior predictive intervals, the solid lines gave the best-fit solution (mean) for the mathematical model, and the colored dots show the experimental datasets. All data were fitted simultaneously. (B) The distribution for the time until reactivation without and with PEP005 treatment calculated from all accepted MCMC parameter estimates is shown in green and blue, respectively. These lengths were significantly shorter with PEP005 treatment than without treatment, as assessed by the repeated bootstrap t test.

Article Snippet: PEP005 (PKC activator) was purchased from Cayman Chemical (Ann Arbor, MI); SAHA (vorinostat; HDAC inhibitor) from Santa Cruz Biotechnology (Dallas, TX); JQ-1 (BRD4 inhibitor) from BioVision (Milpitas, CA); GSK525762A (BRD4 inhibitor) from ChemScene (Monmouth Junction, NJ); Ro5-3335 from Merck (Darmstadt, Germany).

Techniques: Infection, Virus

Journal: Cell Reports Methods

Article Title: A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro

doi: 10.1016/j.crmeth.2021.100122

Figure Lengend Snippet: Mechanisms for the elimination of HIV-1 producible cells in vitro (A) Quantification of intracellular copies of HIV-1 DNA at each time point in experiment 6 ( Figure S2 A). (B) Schematic representation of the individual provirus structures from two different treatment groups and at two time points in experiment 6. Each horizontal bar represents an individual HIV-1 genome, as determined by amplification of near full-length HIV-1 DNA from a single HIV-1 genome and DNA sequencing. The gray bars denote full-length types, and the red bars indicate defective proviruses. (C) Pie charts reflecting the proportion of defective and intact proviruses in experiment 6. (D) Pie charts reflecting the proportion of defective and intact proviruses in PBMCs from three HIV-1-infected individuals. (E) Quantification of intracellular copies of HIV-1 DNA at each time point in experiment 1 ( Figure 3 D). (F) Schematic representation of the individual provirus structures in experiment 1 for the EFdA/PEP005 culture group 17 weeks after drug treatment initiation. (G) Pie chart showing the relative abundance of each HIV-1-infected clone. Chromosomal number and position of each clone are shown in the right panel. (H) Schematic figure of the provirus structure and IS in the expanded clone. A 467-basepair deletion in the 5′ end of 5′ LTR was observed. TSS, transcription start site. Asterisk (∗) represents below detection limit.

Article Snippet: PEP005 (PKC activator) was purchased from Cayman Chemical (Ann Arbor, MI); SAHA (vorinostat; HDAC inhibitor) from Santa Cruz Biotechnology (Dallas, TX); JQ-1 (BRD4 inhibitor) from BioVision (Milpitas, CA); GSK525762A (BRD4 inhibitor) from ChemScene (Monmouth Junction, NJ); Ro5-3335 from Merck (Darmstadt, Germany).

Techniques: In Vitro, Amplification, DNA Sequencing, Infection

Journal: Cell Reports Methods

Article Title: A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro

doi: 10.1016/j.crmeth.2021.100122

Figure Lengend Snippet:

Article Snippet: PEP005 (PKC activator) was purchased from Cayman Chemical (Ann Arbor, MI); SAHA (vorinostat; HDAC inhibitor) from Santa Cruz Biotechnology (Dallas, TX); JQ-1 (BRD4 inhibitor) from BioVision (Milpitas, CA); GSK525762A (BRD4 inhibitor) from ChemScene (Monmouth Junction, NJ); Ro5-3335 from Merck (Darmstadt, Germany).

Techniques: Virus, Infection, Recombinant, Flow Cytometry, Binding Assay, SYBR Green Assay, Purification, Sensitive Assay, Library Quantification, CCK-8 Assay, Software