Human immunodeficiency virus type 1-induced macrophage gene expression includes the p21 gene, a target for viral regulation

Abstract

In contrast to CD4+ T cells, human immunodeficiency virus type 1 (HIV-1)-infected macrophages typically resist cell death, support viral replication, and consequently, may facilitate HIV-1 transmission. To elucidate how the virus commandeers the macrophage's intracellular machinery for its benefit, we analyzed HIV-1-infected human macrophages for virus-induced gene transcription by using multiple parameters, including cDNA expression arrays. HIV-1 infection induced the transcriptional regulation of genes associated with host defense, signal transduction, apoptosis, and the cell cycle, among which the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) gene was the most prominent. p21 mRNA and protein expression followed a bimodal pattern which was initially evident during the early stages of infection, and maximum levels occurred concomitant with active HIV-1 replication. Mechanistically, viral protein R (Vpr) independently regulates p21 expression, consistent with the reduced viral replication and lack of p21 upregulation by a Vpr-negative virus. Moreover, the treatment of macrophages with p21 antisense oligonucleotides or small interfering RNAs reduced HIV-1 infection. In addition, the synthetic triterpenoid and peroxisome proliferator-activated receptor gamma ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which is known to influence p21 expression, suppressed viral replication. These data implicate p21 as a pivotal macrophage facilitator of the viral life cycle. Moreover, regulators of p21, such as CDDO, may provide an interventional approach to modulate HIV-1 replication.

FIG. 1.

Kinetics of HIV-1 infection in monocyte-derived adherent macrophages. (A) Cells were exposed to HIV-1 for the indicated intervals, and mRNAs were extracted and examined by Northern blotting. Bands of 9.1 and 4.3 kb correspond to viral gag/pol and env mRNAs, respectively. (B) Supernatants were collected from infected cultures (days 1 to 15) for p24 ELISA. (C) Cells were incubated for 3 to 10 days after infection and processed for TEM. Original magnification, ×10,000. Ultrastructure analysis revealed detectable virions (C and D) in macrophages by 5 to 7 days postinfection, with increasing virus numbers per cell (C) and numbers of infected macrophages (D) being most evident at or after day 10, as quantified by counting ≥200 cells/time point. The data shown correspond to a representative experiment (n ≥ 4).

FIG. 2.

HIV-1-induced alterations in macrophage transcriptome. The figure shows changes in gene expression in HIV-1-treated macrophages compared to the gene expression levels in mock-infected macrophages from the same donor at intervals, from 0.25 to 14 days (mean values; n = 3 to 6). Increased or decreased gene transcription is represented in red and green, respectively. Genes shown in black indicate no change in transcriptional activity. *, P ≤ 0.05.

FIG. 3.

Increased p21 gene expression in infected macrophages. (A) Kinetic profile of p21 expression from days 0.25 to 14 after infection (n = 3). (B) RPA analysis of uninfected (−) and HIV-1 infected (+) macrophages confirmed the enhanced gene expression for p21, with a minimal effect on p53 (data shown are for a representative donor; n = 2). (C) Densitometric analysis of RPA results for the p21 and p53 genes (shown in panel B), normalized to GAPDH. (D) Macrophages were infected with HIV-1_BAL, the laboratory viral isolate ADA, or the primary isolate 727 and then washed, and the total RNA was collected after 12 days for analysis of p21 transcription by PCR. (E and F) Phytohemagglutinin-blasted T lymphocytes were infected with HIV-1 (IIIB), and day 6 supernatants were examined for p24 Ag. Total mRNA (6 h) was analyzed for p21 transcription by PCR.

FIG. 4.

Infected macrophages express more p21 protein. (A) Overlay confocal images from differential interference contrast (1 and 4) and immunofluorescence labeling for p21 in uninfected (1, 2, and 3) and virus-infected (4, 5, and 6) cells (original magnification, ×400). (B) Fluorescence intensity (FI) analysis (Metamorph) confirmed the enhanced nuclear and cytoplasmic p21 protein, as represented by the signal across equal line segments of nuclear or cytoplasmic areas (data shown are from a representative experiment; n = 3). (C) Increased p21 protein in infected macrophages (12 days) by immunoprecipitation, as quantified by densitometric analysis, relative to that in uninfected cells (n = 3).

FIG. 5.

Induction of p21 gene and protein expression by Vpr. Cells treated with Vpr (6 μg/ml) for 3 h showed increased gene transcription (A) and protein expression (B) for p21. (C) Macrophages were treated with control supernatants from uninfected or mock-transfected cells or from 293T cells infected with the wild-type virus clone pNLAD8 or the pNLAD8 Vpr-negative (#1) or pNLAD8-delta R (#2) R5 macrophage-tropic virus, and 12-day supernatants were analyzed by p24 ELISA. (D) The total RNA was analyzed for p21 and GAPDH by PCR. The data shown are from a representative experiment (n = 2).

FIG. 6.

Inhibition of p21 reduces HIV-1 replication. (A) p21-specific oligonucleotides (1 and 2, 50 nM), but not a control oligonucleotide (3), inhibit HIV-1 growth in replicate cultures, as determined by p24 levels (data for day 12 are shown and are percentages of the positive HIV control with no oligonucleotide). (B) Macrophages treated with p21 siRNA duplexes (5 nM) 5 days prior to HIV infection (% of positive HIV control with no siRNA treatment) (data shown are from a representative experiment; n = 3). Percentages of HIV-1 infection were determined by comparing the p24 levels in untreated versus oligonucleotide- or siRNA-treated macrophages. The inset is a Western blot for p21 from day 14. (C) Cells treated with p21 and negative control siRNAs (5 days) were analyzed by flow cytometry for CD4 and CCR5 cell surface expression. (D) Nested PCR to detect proviral DNA on days 1 and 2 after HIV-1_BaL infection of macrophages treated with p21 siRNA or negative control siRNA. The control represents uninfected cells.

FIG. 7.

Inhibition of HIV-1 replication by CDDO. (A) CDDO-treated cells (as described in Materials and Methods) showed reduced viral replication, as quantitated by p24 levels, compared with dimethyl sulfoxide-treated control cells and untreated cells (day 10) (n = 3; *, P = 0.01 by one-tail paired t test). (B and C) By TEM, reduced numbers of infected cells were observed after CDDO or di-CDDO treatment. Analyses of ≥200 cells/treatment condition revealed the absence or near absence of detectable virions. (D) CDDO-treated cells infected with HIV-1 demonstrated reduced p21 transcription, as determined by RPA (data from day 12 postinfection are shown and are mean values from a representative experiment; n = 2). (E) Terminal deoxynucleotidyl transferase-FITC (apoptotic) and DAPI (nuclear) staining of cultures that were infected with HIV-1 and/or treated with CDDO. (F) Macrophages were infected with HIV-1_BaL or ADA, treated with CDDO (0.1 μM) or left untreated, and analyzed by PCR for p21 and GAPDH. (G) Supernatants (12 days) collected from HIV-1_BaL-, ADA-, or 727-infected cells that were treated with CDDO or left untreated were analyzed for viral replication by p24 ELISA.