Lovastatin Inhibits HIV-1-Induced MHC-I Downregulation by Targeting Nef-AP-1 Complex Formation: A New Strategy to Boost Immune Eradication of HIV-1 Infected Cells

Abstract

Current combined antiretroviral therapy (cART) mainly targets 3 of the 15 HIV proteins leaving many potential viral vulnerabilities unexploited. To purge the HIV-1 latent reservoir, various strategies including "shock and kill" have been developed. A key question is how to restore impaired immune surveillance. HIV-1 protein Nef has long been known to mediate the downregulation of cell-surface MHC-I and assist HIV-1 to evade the immune system. Through high throughput screening of Food and Drug Administration (FDA) approved drugs, we identified lovastatin, a statin drug, to significantly antagonize Nef to downregulate MHC-I, CD4, and SERINC5, and inhibit the intrinsic infectivity of virions. In addition, lovastatin boosted autologous CTLs to eradicate the infected cells and effectively inhibit the subsequent viral rebound in CD4⁺ T-lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Furthermore, we found that lovastatin inhibits Nef-induced MHC-I downregulation by directly binding with Nef and disrupting the Nef-AP-1 complex. These results demonstrate that lovastatin is a promising agent for counteracting Nef-mediated downregulation of MHC-I, CD4, and SERINC5. Lovastatin could potentially be used in the clinic to enhance anti-HIV-1 immune surveillance.

Keywords: AP-1; CD4; HIV-1; MHC-I; Nef; SERINC5; immune surveillance; lovastatin.

Figure 1

Screen and identification of small molecular compounds inhibiting HIV-1 Nef-mediated MHC-I down-regulation. HEK293T cells were transfected with pcDNA3.1-Nef-IRES-GFP or pcDNA3.1-IRES-GFP (800 ng per well), At 48 h post-transfection, Nef-mediated MHC-I down-regulation was analyzed by flow cytometry for staining of MHC-I (A). The ratios of GFP ⁺ MHC-I⁻ HEK293T cells after transfection of pcDNA3.1-Nef-IRES-GFP or pcDNA3.1-IRES-GFP at different concentrations were summarized with bar plot. Data show the means ± standard deviations in three independent experiments (B). Structures of lovastatin and ethionamide (C). Twelve hours after transfection of Nef-I-GFP or empty control plasmid, HEK293T cells were treated with lovastatin or ethionamide at different concentrations. Forty-eight hours after transfection, FACS analysis was performed for the percentages of GFP⁺ MHC-I⁻ cells. Data show the means ± standard deviations in three independent experiments (D).

Figure 2

Lovastatin potently represses the ability of Nef to downregulate MHC-I. Twelve hours after transfection of pcDNA3.1-Nef-IRES-GFP (800 ng per well), HEK293T cells were treated with lovastatin from 0.1 to 12 μM. Forty-eight hours after transfection, FACS analysis was performed for the percentages of GFP⁺ MHC-I⁻ cells (A). The ratios of GFP ⁺ MHC-I⁻ to GFP ⁺ population from pcDNA3.1-Nef-IRES-GFP or pcDNA3.1-IRES-GFP transfections were summarized with bar plot. Data show the means ± standard deviations in three independent experiments (B). The ratios of MHC-I MFI on GFP⁺ to GFP⁻ cells from pcDNA3.1-Nef-IRES-GFP transfections were summarized with bar plot. Data show the means ± standard deviations in three independent experiments (C). The IC50 was calculated according to above data of flow cytometry using GraphPad Prism software (D). Human PBMCs were treated with lovastatin with the given concentrations for 48 h, and the cell viability was then measured by CCK-8 kit. Data show the means ± standard deviations in three independent experiments (E). P-values were calculated using the two tailed unpaired Student's t-test with equal variances, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Lovastatin inhibits the ability of Nef to down-regulate CD4 and SERINC5, and the intrinsic infectivity of virions. TZM-bl cells were transfected with pcDNA3.1-Nef-IRES-GFP or pcDNA3.1-IRES-GFP (800 ng per well). Twelve hours after transfection, the cultures were treated with lovastatin with the given concentrations. Forty-eight hours after transfection, the percentages of the GFP⁺ CD4⁻ cells were analyzed by flow cytometry (A). The ratios of CD4 MFI on GFP⁺ to GFP⁻ cells from pcDNA3.1-Nef-IRES-GFP transfections were summarized with bar plot. Data show the means ± standard deviations in three independent experiments (B). Twelve hours after transfection of SERINC5-GFP/GFP (500 ng per well) and HA-Nef plasmids (500 ng per well), HEK293T cells were treated with lovastatin or vehicle at indicated concentrations, FACS analysis was performed for the percentages of the GFP ⁺ cells at 48 h after transfection (C). The MFI of SERINC5-GFP fusion protein determined by flow cytometry. Data show the means ± standard deviations in three independent experiments (D). Viruses were generated by co-transfection of HIV-1 Env-expressing plasmid and proviral HIV plasmids (pNL4-3-ΔEnv/ΔNef-EGFP or pNL4-3-ΔEnv-EGFP) into SERINC5-expressing or control HEK293T cells with treatment of lovastatin or vehicle. Forty-eight hours after transfection, culture supernatants were harvested. The infectivity of HIV-1 viruses was determined by luciferase assay system in 96-well TZM-bl cell with 10 ng p24-equivalent input viruses (E). P-values were calculated using the two tailed unpaired Student's t-test with equal variances, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Lovastatin restores the MHC-I and CD4 on cell surface after the HIV-1_NL4−3 infection. The activated primary CD4⁺ T cells were infected with HIV-1_NL4−3 or HIV-1_NL4−3ΔNef (p24 titer of 100 ng ml⁻¹). At day 3 post-infection, the cultures were treated with vehicle or lovastatin at 37°C for 48 h. HIV p24 and MHC-I expressions gated on CD3⁺ CD8⁻ subpopulation were analyzed by flow cytometry (A). The percentages of p24⁺ MHC-I⁻ subpopulation and the ratios of MHC-I MFI on GFP⁺ to GFP⁻ cells from the above flow cytometry analysis were summarized with bar plots (B). The percentages of p24⁺ CD4⁻ subpopulation, the ratios of CD4 MFI on GFP⁺ to GFP⁻ cells (C), and the percentages MHC-I⁺ CD4⁺ subpopulation (D) were summarized with bar plots. Data show the means ± standard deviations in three independent experiments. P-values were calculated using the two tailed unpaired Student's t-test with equal variances, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Pre-treatment of lovastatin boosts autologous CTL response against the reactivated latent reservoir from HIV-1 infected individuals. Experimental design. The activated CD4⁺ T cells from HIV-infected individuals receiving suppressive cART were infected with the viruses recovered from the resting CD4⁺ T cells of same patients, CD4⁺ T cells were incubated with lovastatin or vehicle for 48 h, then the cells were washed and mixed with autologous Gag peptides-stimulated CD8⁺ T cells at a 1:1 ratio (A). Every 2 days the cultures were tested for HIV-1 p24 antigen by ELISA (B). Eight days after co-culture, specific killing of infected CD4⁺ T cells by autologous CTLs was determined by LDH assay (C). The residual Gag⁺ T cells gated on CD3⁺ CD8⁻ subpopulation were analyzed by flow cytometry (D). The ratios of residual Gag⁺ to CD3⁺ CD8⁻ subpopulation were summarized from the above flow cytometry analysis (E). Data show the means ± standard deviations from results of three HIV-1 infected individuals. P-values were calculated using the two tailed paired Student's t-test with equal variances, n = 3. *p < 0.05.

Figure 6

Restoration of MHC-I by lovastatin is independent from mevalonate pathway. HEK293T cells were treated with lovastatin from 2 to 20 μM. Forty-eight hours later, ELISA analysis the concentration of the cholesterol in the drug-treated cells (A). Structures of lovastatin, simvastatin and fluvastatin (B). Twelve hours after transfection of pcDNA3.1-Nef-IRES-GFP or pcDNA3.1-IRES-GFP (800 ng per well), HEK293T cells were treated with lovastatin, simvastatin, fluvastatin or bisphosphonates (zoledronic acid and pamidronate). Forty-eight hours after transfection, the ratios of GFP ⁺ MHC-I⁻ to GFP ⁺ population were analyzed by flow cytometry and summarized with bar plot. Data show the means ± standard deviations in three independent experiments (C). The ratios of MHC-I MFI on GFP⁺ to GFP⁻ cells from pcDNA3.1-Nef-IRES-GFP transfections was determined by flow cytometry. Data show the means ± standard deviations in three independent experiments (D). P-values were calculated using the two tailed unpaired Student's t-test with equal variances, n = 3. **p < 0.01, ***p < 0.001.

Figure 7

Lovastatin directly targets Nef and inhibits the interaction between Nef and AP-1. Molecular docking studies were performed to explore the binding modes of lovastatin or its hydroxy acid form with the crystal structure of Nef in complex with of AP-1 μ1 subunit and MHC-I cytoplasmic domain (PDB:4emz), the B and C chains in green belong to HIV Nef, the A and M chains in red belong to AP-1, and the D and E chains in purple belongs to MHC-I (A). The pose of lovastatin or lovastatin hydroxy acid in binding pocket, the pharmacophore model, and the specific binding details of the interaction between ligand with Nef–AP-1 complex: the carbon backbone of lovastatin is in yellow, the Glu63 and Phe68 of chain B carbon backbone are in green and the Lys302 of chain A carbon backbones is in red (B). Twelve hours after transfection of Nef-IRES-GFP (800 ng per well), HEK293T cells were treated with hydroxy acid of lovastatin at 4 μM. Forty-eight hours after transfection, MHC-I density on cell surface was analyzed by flow cytometry. These data represent three independent experiments (C). HA-tagged Nef or GFP constructs were transfected into HEK293T cells. Twelve hours after transfection, cells were treated with lovastatin or vehicle. Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-HA antibody, and subsequently detected with anti-HA, anti-AP1 or anti-GAPDH antibody. HA-GFP as IP control, GAPDH was used as a loading control. Graph shows normalized levels of HA-Nef– or HA-GFP–interacted AP1 (D). HA-tagged wild type Nef or mutant constructs were transfected into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-HA antibody, and subsequently detected with anti-HA, anti-AP1 or anti-GAPDH antibody. GAPDH was used as a loading control. Graph shows normalized levels of HA-Nef–interacted AP1 (E). Surface plasmon resonance experiments were performed to measure the binding affinity of lovastatin on Nef or Nef ^E63A/F68A and fluvastatin on Nef with a BIAcore T100 Biosensor System. These data represent two independent experiments (F). P-values were calculated using the two tailed unpaired Student's t-test with equal variances, n = 3. *p < 0.05, **p < 0.01.