KAT5 depletion prevents HIV from efficiently establishing latency.

<p><b>A.</b> A schematic diagram showing the procedure and timeline of experiments to progressively establish latency in Jurkat cell pools stably expressing either shKAT5 or shScramble. d.p.i.: Days post infection. <b>B.</b> Lysates of the cell pools expressing the indicated shRNA were examined by Western blotting for the proteins labeled on the left. <b>C.</b> The GFP(+) cells that were selected by FACS on 2 d.p.i from each cell pool were checked by flow cytometry for the percentages of GFP(+) cells on the indicated d.p.i. and the average of three measurements at each time point is shown. <b>D.</b> Representative immunofluorescence flow cytometry analysis of shScramble- and shKAT5-expressing cells that were either uninfected or infected with HIV and harvested at the indicated d.p.i.</p

Antagonizing KAT5 but not KAT7 or KAT8 reverses HIV latency and potentiates conventional LRAs.

<p><b>A. D.</b> & <b>F.</b> The Jurkat 2D10-based inducible CRISPRi-KAT5-sg1, CRISPRi-KAT7-sg1, and CRISPRi-KAT8 cells were treated with (+) or without (-) doxycycline (Dox) and analyzed by RT-qPCR for the KAT5/7/8 mRNA levels, which were normalized to those of ActB, and by Western blotting for the indicated proteins. <b>B. E.</b> & <b>G.</b> CRISPRi-KAT5-sg1, CRISPRi-KAT7-sg1, and CRISPRi-KAT8 cells were treated with or without Dox (1 μl/ml) and the various LRAs at the indicated concentrations. The treated cells were subjected to FACS analysis to determine the percentage of GFP(+) cells in each cell population. <b>C.</b> Wild-type 2D10 cells were treated with combinations of MG-149 and DMSO or the various LRAs at the indicated concentrations and subjected to FACS analysis as in B. Each column in all panels represents the average of three independent measurements, with the error bars indicating mean +/- SD.</p

The KAT5-Acetyl-Histone4-Brd4 axis silences HIV-1 transcription and promotes viral latency

<div><p>The bromodomain protein Brd4 promotes HIV-1 latency by competitively inhibiting P-TEFb-mediated transcription induced by the virus-encoded Tat protein. Brd4 is recruited to the HIV LTR by interactions with acetyl-histones3 (AcH3) and AcH4. However, the precise modification pattern that it reads and the writer for generating this pattern are unknown. By examining a pool of latently infected proviruses with diverse integration sites, we found that the LTR characteristically has low AcH3 but high AcH4 content. This unusual acetylation profile attracts Brd4 to suppress the interaction of Tat with the host super elongation complex (SEC) that is essential for productive HIV transcription and latency reversal. KAT5 (lysine acetyltransferase 5), but not its paralogs KAT7 and KAT8, is found to promote HIV latency through acetylating H4 on the provirus. Antagonizing KAT5 removes AcH4 and Brd4 from the LTR, enhances the SEC loading, and reverses as well as delays, the establishment of latency. The pro-latency effect of KAT5 is confirmed in a primary CD4+ T cell latency model as well as cells from ART-treated patients. Our data thus indicate the KAT5-AcH4-Brd4 axis as a key regulator of latency and a potential therapeutic target to reactivate latent HIV reservoirs for eradication.</p></div

Inhibition of KAT5 in a primary cell latency model and ART-suppressed patient cells enhances HIV latency reversal and virion release.

<p><b>A.</b> Scheme showing the procedure followed to generate latently infected resting Th17 cells. <b>B.</b> Memory CD4+ T cells from a healthy donor were pretreated with 1 μM SAHA overnight and then incubated with the indicated concentrations of MG-149 for 24 hr. Western blotting analyses were conducted to detect the levels of AcH4, AcH3, and total H4 in whole cell lysates. The AcH4:AcH3 ratio in each lane was measured by densitometry and shown on the right. <b>C.</b> Representative immunofluorescence flow cytometry analysis of Nef-Cy5 and CD8a-EGFP expression in latently infected primary Th17 cells that were either TCR-activated or treated with 1 μM JQ1 for 48 hr in the presence or absence of 10 μM MG-149. EGFP reporter used for positive selection remained high in the latently infected cells due to prolonged stability of the membrane-bound CD8a-EGFP fusion protein. <b>D.</b> Representative immunofluorescence flow cytometry analysis of Nef-Cy5 and CD8a-EGFP expression in latently infected primary Th17 cells that were either untreated or treated with 5 μM JQ1 with or without MG-149 for 48 hr. <b>E.</b> Primary cells from ART-suppressed HIV-1-infected individuals were treated with the indicated drug(s) for 24 hr. HIV-1 RNA in culture supernatant were quantified with the Roche COBAS AmpliPrep/TaqMan HIV-1 Qualitative Test system. The viral copy numbers were presented as fold induction relative to the DMSO control. Numbers in parentheses indicate number of individuals used for each treatment. N.S. not significant; *P < 0.05; **P < 0.01. Error bars represent standard error of the mean (SEM). <b>F.</b> Diagram depicting KAT5 but not KAT7 and KAT8 as a host factor for suppressing HIV proviral transcription and promoting viral latency. See text for more details.</p

Phosphorylation of CDK9 at Ser175 Enhances HIV Transcription and Is a Marker of Activated P-TEFb in CD4⁺ T Lymphocytes

<div><p>The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.</p></div

Antagonizing KAT5 activates Tat-dependent HIV transcription but inhibits cellular primary response genes.

<p><b>A.</b> & <b>B.</b> Jurkat 2D10-based CRISPRi-KAT5-sg1 (A) and parental 2D10 (B) cells were treated with (+) or without (-) Dox (A) or MG-149 (B). The mRNA levels of the indicated genes were measured by RT-qPCR and normalized to those of ActB and shown as averages of three independent measurements. The error bars indicate mean +/- SD and the asterisks denote levels of statistical significance calculated by two-tailed Student’s <i>t</i>-test (*: p<0.05, **: p<0.01, and ***: p<0.001). <b>C.</b> & <b>D.</b> Luciferase activities were measured in extracts of Jurkat 1G5 or Jurkat 1G5+Tat cells containing an integrated HIV-1 LTR-luciferase reporter construct and stably expressing the indicated shRNA (C) or treated with the indicated concentrations of MG-149 (D). <b>E.</b> Luciferase activities were measured in extracts of 293T cells that were transfected with the HIV-1 LTR-luciferase reporter construct together with the vector expressing FLAG-KAT5- and/or Tat-HA as indicated. All experiments in C to E were performed in triplicates with the error bars representing mean +/- SD and the activities in the first column set to 1.0. An aliquot of each cell extract was examined by Western blotting for the proteins labeled on the left.</p

CDK9 kinase activity does not require S175.

<p>Radioactive <i>in vitro</i> kinase assays perfomed using FLAG-CDK9 complexes isolated from Jurkat 2D10 cells stimulated for 20 h with 10 ng/mL TNF-α to induce Tat expression. Western blots of these IPs and the corresponding WCEs are shown in the lower panel of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat-1003338-g005" target="_blank"><b>Fig. 5B</b></a>. The kinase assays were performed in the absence (A) or presence (B) of 250 ng of His-tagged full length human pol II CTD repeat substrate. Inhibition of activity by pretreatment with 100 nM flavopiridol confirms the activity in both assays to be CDK9 kinase. Note that the S175A and S175D mutations display wildtype kinase activity in both assays.</p

Modeling and Simulation Based Analysis of Multi-Class Traffic with Look-Ahead Controlled Vehicles

Additional file 7: Fig. S7. TLR and PTEF-b inhibition impairs TLR-mediated HIV reactivation. a TLR ligands reactivate HIV in an NF-κB-dependent manner. Treatment of THP-1/HIV (HA3) cells with TNF-α (10 ng/mL) or TLR ligands (Pam3CSK4 at 0.1 µg/mL, HKLM at 108 cells/mL, poly (I:C) at 10 µg/mL, LPS at 1 µ/mL, flagellin at 1 µ/mL, FSL-1 at 1 µg/mL, imiquimod at 10 µg/mL, ssRNA40 at 5 µg/mL, and ODN2006 at 5 µM) for 16 h after a 2-h pre-incubation with either 100 µM of IKKγ NEMO binding domain inhibitory peptide (red bars; Inh Pep) or equivalent amount of the control peptide (blue bars; Imgenex) (X-axis). Y-axis represents % of GFP-expressing cells after FACS measurements and blue squares % of viable cells after PI exclusion quantification (right Y-axis). Error bars depict the standard deviation of three different experiments. b Partial inhibition of TNF-α-, IL-1β-, or TLR-mediated HIV reactivation by P-TEFb inhibitors. Human hµglia/HIV (HC01) and (HC69), and rat hT-CHME-5/HIV (HC03) and (HC14) microglial cells were untreated (black) or pre-treated with DRB (red; 10 µM) or flavopiridol (blue; 30 nM) for 30 min prior to treatment with TNF-α (30 ng/mL), IL-1β (10 pg/mL), LPS (1 µg/mL), or poly (I:C) (10 µg/mL), as shown in the X-axis, for 16 h prior to quantification of GFP (Y-axis)

Post-translational modifications (PTMs) of CDK9 Isoform 2 (117 amino acid extension at N-Terminus) identified by tandem mass spectrometry analysis.

<p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of the CDK9 isoform 2 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

Ser175 is not required for P-TEFb formation and the assembly of the 7SK snRNP complex.

<p>(A) FLAG-CDK9 wildtype, S175A, S175D, T186A, or T186D were stably expressed in Jurkat 2D10 cells using the MSCV retroviral expression system. Western blotting analysis was performed on the whole cell extracts (left panels) and the corresponding anti-FLAG-CDK9 immunoprecipitates (right panels) using antibodies against CycT1, CDK9 and the 7SK RNP protein components HEXIM1 and LARP7. (B) Quantitative analysis of protein levels. Protein concentrations were estimated by densitometry of the Western blots and normalized against total immunoprecipitated CDK9. The data are from five independent experiments. Error bars: ± standard error of the mean.</p