A limited group of class I histone deacetylases acts to repress human immunodeficiency virus type 1 expression

Abstract

Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4(+) T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4(+) T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4(+) T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.

Figures

FIG. 1.

HDAC mRNA expression in resting CD4+ T cells from aviremic, HIV-1-positive patients. (A) HDAC1, -3, and -7 are the most highly expressed HDAC mRNAs in resting CD4+ T cells. Microarray analysis of mRNA expression in the resting CD4+ T cells from three HIV-1-positive patients provided relative intensities of HDAC mRNA expression. (B) HDAC1 to -11 are detectable in resting CD4+ T cells. Whole-cell extracts were obtained from the resting CD4+ T cells of HIV-1-positive patients, and 20 μg of protein was subjected to Western blot analysis with antibodies specific for HDAC1 to -11. An antibody against the nuclear envelope marker lamin B1 was used as a loading control. As a positive control (+) for the anti-HDAC10 antibody, 2 μg of whole-cell extracts from 293T cells transfected with an HDAC10 expression plasmid was subjected to Western blotting. Although HDAC10 was not detected in patient extracts in the experiment shown, it was detected when 40 μg of extracts was assayed (data not shown).

FIG. 2.

HDAC5, -8, and -11 are excluded from the nuclei of resting CD4+ T cells. Resting CD4+ T cells from an aviremic, HIV-1-positive patient (patient 5 from Fig. 1A) were maintained in medium (Rest) or activated by incubation with 1 μg/ml of the mitogen PHA overnight (Act) before cellular lysates were harvested. Proteins were separated into nuclear (N) and cytoplasmic (C) fractions, and 15 μg of extracts was probed with antibodies targeting HDAC1 to -11 in Western blot analysis. Antibodies against lamin B1 and glyceraldehyde-3-phosphate dehydrogenase were used to assess loading of nuclear and cytoplasmic lysates. HDAC5, -8, and -11 were primarily localized to the cytoplasm in CD4+ T cells. Following T-cell activation with PHA, HDAC7 expression increased and became sequestered in the cytoplasm, while HDAC11 expression decreased.

FIG. 3.

HDAC1, -2, and -3 are recruited to the HIV-1 LTR in the J89GFP cell line model of latency. (A) HDAC localization in J89GFP cells is similar to that in resting CD4+ T cells. Nuclear (N) and cytoplasmic (C) protein extracts (30 μg each) from J89GFP cells were probed with antibodies against HDAC1 to -11 in Western blot analysis. Antibodies targeting lamin B1 and alpha-tubulin were used as loading controls for nuclear and cytoplasmic extracts, respectively. (B) HDAC1, -2, and -3 associate with the HIV-1 LTR. Antibodies targeting the nuclear class I HDACs HDAC1, -2, and -3 were used in ChIP assays in J89GFP cells. Rabbit IgG serum was used to assay nonspecific immunoprecipitation of LTR DNA. (C) The nuclear class II HDACs HDAC4, -6, and -7 do not associate with the HIV-1 LTR. ChIP assays were performed in J89GFP cells. HDAC2 was used as a positive control, and rabbit IgG serum was used as a negative control. Values in panels B and C represent the enrichment of LTR DNA over the IgG negative control as determined by quantitative PCR. Experiments were performed on at least three occasions. Data are expressed as the means ± standard errors of the means.

FIG. 4.

HDAC2 and HDAC3 negatively regulate the HIV-1 LTR. (A) siRNA-mediated knockdown of HDAC2 induced a significant increase in LTR-driven lacZ expression compared to the mock control (n = 64; ***, P < 0.001). P4/R5 cells were transfected with siRNAs targeting the class I HDACs HDAC1, -2, -3, and -8. Twenty hours posttransfection, cells were incubated with 1% DMSO (vehicle control) for an additional 20 h. As a control, cells were transfected with nonspecific (NS) siRNA. Values are displayed in relative light units and were derived from Gal-Screen assays of cellular lysates. (B) siRNA knockdown of HDAC3 in conjunction with exposure to a submaximal concentration of the HDAC inhibitor TSA upregulated LTR-driven lacZ expression compared to cells that were mock transfected and exposed to TSA (n = 32; ***, P < 0.001). Cells were treated as for panel A except that 1 μM TSA was added in place of DMSO. (C) There were no differences in cell proliferation following HDAC knockdown compared to the mock controls (n = 12, P = 0.522 for DMSO experiment; n = 12, P = 0.307 for TSA experiment). alamarBlue assays were used to assess cell viability in panels A and B. Data in panels A to C are the combined results of at least three experiments and are expressed as the means ± standard errors of the means.