Fig 1: Increased cellular Hsp90 activity accelerates HIV transcription in persistently infected cells. A, nitric-oxide synthase (NOS) activity in 8E5/LAV cells at 37 and 39.5 °C with thapsigargin (Thaps.) as a positive control at 37 °C. Data represent mean ± S.E. (n = 3). B, quantitative fluorescence Western blotting analysis of 8E5/LAV cell lysates. Results shown are representative of four independent experiments. C, antiviral activity of 17-AAG in 8E5/LAV cells at 37 and 39.5 °C. Mean cytotoxicity at 37 °C (5.2 μm) and 39.5 °C (5.2 μm) is shown. D, antiviral activity of 17-AAG on 8E5/LAV cells pretreated with IUdR at 37 and 39.5 °C. Mean cytotoxicity at 37 °C (5.6 μm) and 39.5 °C (5.3 μm) is shown. E, cell-associated HIV RNA from 8E5/LAV cells pretreated with IUdR at 37 and 39.5 °C. p = 0.26 at 100 nm 17-AAG and p = 0.34 at 32 nm 17-AAG by Student's t test. Data represent mean ± S.E. (error bars) (n = 3).
Fig 2: Hsp90 controls HIV transcription. Shown is the Dual-Glo luciferase assay at 37 and 39.5 °C and at 39.5 °C with 17-AAG in HIV-infected 8E5/LAV cells (left) and in uninfected Jurkat T cells (right). Data represent mean ± S.E. (error bars) (n = 4). *, p < 0.0001 by Student's t test comparing luciferase expression at 39.5 °C with luciferase expression at 39.5 °C in the presence of 1 µm 17-AAG.
Fig 3: Long lasting effect of Hsp90 inhibition. A, PBMC pretreated with 1 µm 17-AAG for 24 h were inoculated with HIV at multiplicity of infection of 0.001 and 0.1 from 1 to 6 days after drug treatment. Supernatant p24 was estimated after 7 days of culture. B, uninfected PBMC pretreated with 1 µm 17-AAG for 24 h were tested for cytotoxicity from 1 to 6 days after drug treatment. Data represent mean ± S.E. (error bars) (n = 4).
Fig 4: Dramatically reduced HIV DNA copy number in circulating PBMC from Hsp90 inhibitor-treated mice yet minimal reduction of HIV DNA in spleen cells with decreased numbers of HIV RNA-positive cells. A, cell-associated HIV DNA copy number was estimated on total DNA extracted from NSG-BLT mouse PBMC and spleen cells. Open circles, mice that were not injected with CD34+ hematopoietic stem/progenitor cells. The HIV DNA copy number is compared with that of human CCR5 DNA using PCR primers specific to human CCR5. B, HIV RNA (coding RNA+, fuchsia) was detected by RNAscope using a HIV gag-pol probe in formaldehyde-fixed frozen spleen tissue samples (obtained 11 weeks after treatment cessation from mice shown in A). The RNAscope assay was followed by colorimetric immunohistochemistry for p24 (brown), and nuclei were counterstained with hematoxylin. Images were acquired at ×630 magnification, and HIV RNA-positive cells were digitally quantified by ImageJ software analysis. The total number of HIV RNA-positive cells (abundantly expressing HIV RNA or containing a single HIV RNA transcript) was quantified using 34 images from three spleens (untreated group), 38 images from four spleens (EFdA only), 37 images from four spleens (EFdA plus 17-AAG), and 33 images from four spleens (EFdA plus AUY922). In the HIV RNA+ bar graph, p = 0.0071 (**) by Mann-Whitney U test.
Fig 5: Hsp90 inhibition reduced the persistent tissue reservoir. A, repeat experiment with AUY922, a second-generation Hsp90 inhibitor. Groups of viremic HIVJR-CSF-infected NSG-BLT mice were treated orally once a day with 10 mg/kg/day EFdA only for 4 weeks or with EFdA plus twice-daily subcutaneous injection of 17-AAG or AUY922 (10 mg/kg/day) for the last 2 weeks. The treatment period is indicated by shading, and all mice were from the same NSG BLT mouse cohort. Mouse peripheral blood human CD4+ T cell counts at the time of treatment cessation were highly similar between groups with means of 130 CD4+ T cells/ μl (untreated), 110 CD4+ T cells/μl (EFdA only), 94 CD4+ T cells/μl (EFdA plus 17-AAG), and 66 CD4+ T cells/μl (EFdA plus AUY922). Open circles, implanted mice in the cohort that were not injected with CD34+ hematopoietic stem/progenitor cells. p values for Fisher's exact test are shown. B, IUPM assay on terminal PBMC and spleen cells (obtained at both 6 and 11 weeks after treatment cessation from mice shown in A) at 37 and 39.5 °C and in the presence of 10 nm SAHA. Serial dilutions of mouse-derived cells were cocultivated with PHA-stimulated PMBC for 7 days, and IUPM were determined by detection of p24 in the supernatants.
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