Heat shock protein 90 controls HIV-1 reactivation from latency

Abstract

Latency allows HIV-1 to persist in long-lived cellular reservoirs, preventing virus eradication. We have previously shown that the heat shock protein 90 (Hsp90) is required for HIV-1 gene expression and mediates greater HIV-1 replication in conditions of hyperthermia. Here we report that specific inhibitors of Hsp90 such as 17-(N-allylamino)-17-demethoxygeldanamycin and AUY922 prevent HIV-1 reactivation in CD4+ T cells. A single modification at position 19 in the Hsp90 inhibitors abolished this activity, supporting the specificity of the target. We tested the impact of Hsp90 on known pathways involved in HIV-1 reactivation from latency; they include protein kinase Cs(PKCs), mitogen activated protein kinase/extracellular signal regulated kinase/positive transcriptional elongation factor-b and NF-κB. We found that Hsp90 was required downstream of PKCs and was not required for mitogen activated protein kinase activation. Inhibition of Hsp90 reduced degradation of IkBα and blocked nuclear translocation of transcription factor p65/p50, suppressing the NF-κB pathway. Coimmunoprecipitation experiments showed that Hsp90 interacts with inhibitor of nuclear factor kappa-B kinase (IKK) together with cochaperone Cdc37, which is critical for the activity of several kinases. Targeting of Hsp90 by AUY922 dissociated Cdc37 from the complex. Therefore, Hsp90 controls HIV-1 reactivation from latency by keeping the IKK complex functional and thus connects T-cell activation with HIV-1 replication. AUY922 is in phase II clinical trial and, in combination with a PKC-ϑ inhibitor in phase II clinical trial, almost completely suppressed HIV-1 reactivation at 15 nM with no cytotoxicity. Selective targeting of the Hsp90/Cdc37 interaction may provide a powerful approach to suppress HIV-1 reactivation from latency.

Fig. 1.

Ansamycin antibiotics repress HIV-1 reactivation from latency. (A) Crystal structure of GA bound to the N-terminal ATPase pocket of Hsp90 (Protein Data Bank: 1YET). (B) J-Lat cells (A2 clone) were stimulated with 5 nM TPA for 24 h to induce HIV-1 reactivation in the presence of the indicated concentrations of 17-AAG and analyzed by FACS; ctr−, DMSO only. Bars show average values ± SD, n = 3. (C) Same as B, but J-Lat clones 9.2 and 10.6, harboring a full-length HIV-1 provirus, were used. Bars show average values less background (no TPA) ± SD, n = 3.

Fig. 2.

Substitutions at position 19 reduce potency of Hsp90 inhibitors that repress HIV-1 reactivation. Chemical structures of different Hsp90 inhibitors are shown, with the specific substitutions at position 19 highlighted (red circles). The IC₅₀ is indicated below each compound structure. IC₅₀ was calculated in J-Lat A2 cells by ExcelFit as the concentration of drug, reducing by 50% the MFI values relative to cells treated with TPA only.

Fig. 3.

Phorbol esters induce several pathways regulating HIV-1 reactivation. (A) Schematic representation of the pathways activated by TPA or prostratin (PS) leading to HIV-1 reactivation. (B) J-Lat cells (A2 clone) were stimulated with 5 nM TPA for 24 h to induce HIV-1 reactivation in the presence of the indicated concentrations of 17-AAG or SAHA or a combination of the two and analyzed by FACS; ctr−, DMSO only. Bars show average values ± SD, n = 3.

Fig. 4.

The MEK/MAPK pathway and PKCs are not targeted by Hsp90 inhibitors. (A) J-Lat cells (A2 clone) were stimulated with 10 nM TPA for 24 h to induce HIV-1 reactivation in the presence of 17-AAG (2 μM) and U0126 (20 μM) and analyzed by FACS. Bars show average values ± SD, n = 3. (B) Cells were stimulated with TPA (10 nM) for 15 min in the presence of the indicated concentrations of U0126. Samples were collected and analyzed by Western blot to detect phosphorylated p42/p44 MAPK. (C) Cells were stimulated with TPA (10 nM) for 15 or 30 min in the presence of 2 μM 17-AAG and analyzed by Western blot as above. (D) J-Lat cells (A2 clone) were stimulated with 10 nM TPA for 24 h to induce HIV-1 reactivation in the presence of 17-AAG (2 μM) and the indicated concentrations of Go or Gx. Bars show average values ± SD, n = 3. (E) Cells were stimulated with TPA (10 nM) for 15 or 30 min in the presence of 17-AAG (2 μM) and analyzed by Western blots with an anti-pan phosphorylated PKC antibody.

Fig. 5.

Hsp90 inhibitors target the NF-κB pathway to block HIV-1 reactivation from latency. (A) J-Lat cells (A2 clone) were stimulated with TNFα (5 ng/mL) or TPA (5 nM) for 24 h to induce HIV-1 reactivation. (B) Cells stimulated with TNFα (5 ng/mL) or (C) TPA (5 nM) for 24 h in the presence of the indicated concentrations of AUY922 or 17-AAG were analyzed by flow cytometry. Bars in A–C show average values ± SD, n = 3. (D) Cells were stimulated with TPA (5 nM) for 15 or 30 min in the presence of 17-AAG (2 μM) and analyzed by Western blot with an antibody against IkBα. (E) Image-J quantification of IkBα band intensity relative to actin from three independent Western blots. Bars show average values ± SD, n = 3; control, no TPA, no 17-AAG.

Fig. 6.

Hsp90 inhibitors act on the NF-κB pathway. (A) J-Lat cells were nucleofected with a 1:2 combination of a plasmid encoding mCherry and a plasmid encoding an hyperactive IKK mutant (mut IKKβ S177E, S181E) or the same total amount of mCherry plasmid only (Ctr). Twenty-four hours after nucleofection, cells were analyzed by two color FACS to detect the level of HIV-1 reactivation (GFP channel) within the mCherry+ cell population. Nucleofection of mut IKKβ S177E, S181E plasmid induced HIV-1 reactivation, which was repressed by 17-AAG (2 μM). (B) U2OS_exo cells were incubated with AUY922 for 20 h at the indicated concentrations before stimulation with TNFα (30 ng/mL) for 30 min and immunostaining to detect NF-κB (p65). Nuclear and cytoplasmic localization were scored by manual counting of at least 350 cells in triplicate. DMSO 1%, cells stimulated by TNFα in the absence of AUY922. (C) Representative pictures used to quantify nuclear and cytoplasmic localization of NF-κB.

Fig. 7.

AUY922 dissociates Cdc37 from the Hsp90/IKK complex. (A) J-Lat cells were stimulated with TPA (10 nM) for 15 min and cell extracts used to perform immunoprecipitations with an anti-IKKγ polyclonal antibody or preimmune serum in the presence of 4 μM AUY922 or 4 μM 19-Me-GA. Samples were analyzed by Western blot with monoclonal antibodies against IKKγ, Hsp90, and Cdc37. (B) Image-J quantification of the Cdc37 band intensity detected in the supernatant and the pellet fractions. Each bar represents an individual experiment.

Fig. 8.

Combining AUY922 and sotrastaurin to repress HIV-1 reactivation. J-Lat A2 cells were stimulated by TPA (10 nM) for 24 h in the presence of the indicated concentrations of AUY922 and sotrastaurin, alone or in combination. Samples were analyzed by FACS and data plotted using MacSynergy II software. (A) Representative McSynergy II plot: areas of the graph above zero indicate an additive or synergistic effect. (B) Representative checkerboard grid used to calculate the plot shown in A. (C) Average McSynergy II score in μM²% of four independent experiments at 95% confidence interval. Values between 25 and 50 μM²% with log volumes of >2 and <5 indicate modest but significant synergy (–52).