Retinoid-induced repression of human immunodeficiency virus type 1 core promoter activity inhibits virus replication

Abstract

The rates of mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), progression to AIDS following HIV-1 infection, and AIDS-associated mortality are all inversely correlated with serum vitamin A levels (R. D. Semba, W. T. Caiaffa, N. M. H. Graham, S. Cohn, and D. Vlahov, J. Infect. Dis. 171:1196-1202, 1995; R. D. Semba, N. M. H. Graham, W. T. Caiaffa, J. B. Margolik, L. Clement, and D. Vlahov, Arch. Intern. Med. 153:2149-2154, 1993; R. D. Semba, P. G. Miotti, J. D. Chiphangwi, A. J. Saah, J. K. Canner, G. A. Dallabetta, and D. R. Hoover, Lancet 343:1593-1596, 1994). Here we show that physiological concentrations of vitamin A, as retinol or as its metabolite, all-trans retinoic acid, repressed HIV-1Ba-L replication in monocyte-derived macrophages (MDMs). Repression required retinoid treatment of peripheral monocytes during their in vitro differentiation into MDMs. Retinoids had no repressive effect if they were added after virus infection. Retinol, as well as all-trans retinoic acid and 9-cis retinoic acid, also repressed HIV-1 long terminal repeat (LTR)-directed expression up to 200-fold in transfected THP-1 monocytes. Analysis of HIV-1 LTR deletion mutants demonstrated that retinoids were able to repress activation of HIV-1 expression by both NF-kappaB and Tat. A cis-acting sequence required for retinoid-mediated repression of HIV-1 transcription was localized between nucleotides -51 and +12 of the HIV-1 LTR within the core promoter. Protein-DNA cross-linking experiments identified four proteins specific to retinoid-treated cells that bound to the core promoter. We conclude that retinoids render macrophages resistant to virus replication by modulating the interaction of cellular transcription factors with the viral core promoter.

FIG. 1

Retinol and RA repress HIV-1 replication in MDMs. MDMs were grown either with or without retinoids for 5 days and then infected with HIV-1_Ba-L, a macrophage-tropic virus isolate. After infection, retinoid treatments were continued in those cultures that were pretreated and begun in other cultures that were previously untreated (post-infection). Some cultures were left untreated and served as controls. (a) Virus production in untreated and RA-pretreated (10⁻⁹ M) MDMs was measured as cell-free reverse transcriptase activity. Each time point represents the average (± standard deviation) of three independent infections. (b) Levels of virus production 15 days after infection. Each sample represents the average (± standard deviation) of three independent infections. untr., untreated.

FIG. 2

Retinol and its metabolic derivatives RA and 9cRA repress HIV-1 LTR-directed expression in THP-1 monocytes. (a) Untreated or RA-treated (10⁻⁷ M; 4 days) THP-1 monocytes were transfected with p(−139/+84)CAT, a plasmid containing a portion of the HIV-1 LTR (nucleotides −139 through +84) directing the expression of CAT. Some transfections also contained pCMVcTat (Tat), a plasmid expressing Tat. Levels of CAT activity were measured 16 to 18 h after transfection. The data are the averages (± standard deviations) of four independent transfections. Fold repression was calculated as the ratio of CAT activities measured from untreated cells to those in retinoid-treated cells. +, treated; −, untreated. (b) Untreated (−) or retinoid-treated (retinol [ROL], 10⁻⁶ M, 4 days; RA [AT], 10⁻⁷ M, 4 days; 9cRA [9c], 10⁻⁷ M, 4 days) THP-1 cells were cotransfected with p(−139/+84)CAT and pCMVcTat. Levels of CAT activity were measured 16 to 18 h after transfection. The data are the averages (± standard deviations) of three independent transfections. trt, treatment.

FIG. 3

Time course of RA-mediated repression. (a) THP-1 monocytic cells were treated for the indicated times with 10⁻⁷ M RA. Both treated and untreated cells were then cotransfected with p(−139/+84)CAT and pCMVcTat. Levels of CAT activity were measured 16 to 18 h after transfection. The data obtained from two independent experiments are shown. (b) THP-1 monocytes were treated as described above and then transfected with pCMVCAT containing the CMV immediate-early promoter directing CAT expression. Levels of CAT activity were measured 16 to 18 h after transfection. The data obtained from two independent experiments are shown.

FIG. 4

Concentration dependence of retinoid-mediated repression. THP-1 monocytic cells were treated for 4 days with the indicated concentration of either RA (solid symbols) or retinol (open symbols) and then cotransfected with p(−139/+84)CAT and pCMVcTat. Levels of CAT activity were measured 16 to 18 h after transfection. The data obtained from two independent experiments are shown. Circles, experiment 1; squares, experiment 2.

FIG. 5

RA-mediated repression and THP-1 differentiation are not synonymous. Untreated or RA-treated (10⁻⁷ M; 4 days) THP-1 monocytes were cotransfected with p(−139/+84)CAT and pCMVcTat. One-half of the transfected cells were also treated with 50 nM PMA. Levels of CAT activity were measured 24 h after transfection. The data are the averages (± standard deviations) of four independent experiments. +, treated; −, untreated.

FIG. 6

RA represses HIV-1 expression from the core promoter. Untreated (−; shaded bars) or RA-treated (10⁻⁷ M; 4 days) (+; solid bars) THP-1 cells were transfected with HIV-1 LTR-CAT reporter plasmids containing different portions of the HIV-1 promoter. p(−86/+84)CAT includes the binding sites for Sp1 and TFIID and encodes TAR, the binding site for Tat. p(−51/+84)CAT includes the binding site for TFIID and encodes TAR. p(−86/+12)CAT includes the binding sites for Sp1 and TFIID but does not encode TAR. All transfections also contained pCMVcTat. Levels of CAT activity were measured 16 to 18 h after transfection. The data are the averages (± standard deviations) of at least three to four independent transfections.

FIG. 7

Retinoid treatment of THP-1 monocytes alters the pattern of factors which bind to the HIV-1 promoter. (a and b) Cellular extracts were prepared from both untreated THP-1 monocytes (−) and cells that were treated for 4 days with 10⁻⁷ M RA (+). Extracts were incubated with radioactive DNA probes corresponding to either nucleotides −51 through +84 (a) or −51 through +12 (b) of the HIV-1 promoter. Protein-DNA complexes were resolved on a nondenaturing polyacrylamide gel. Extracts from untreated cells formed a complex, B2, with the −51-through-+84 probe. A complex with a slower electrophoretic mobility, B2RA, was formed with extracts from RA-treated cells. Both complexes were specifically competed for by a 100-fold molar excess of unlabeled self DNA (comp) but not by a fragment containing the CMV immediate-early promoter (data not shown). Similar complexes were formed when the −51-through-+12 probe was used. (c) Cellular extracts were prepared from either untreated THP-1 monocytes (−) or cells treated for 4 days with either 10⁻⁷ M RA, 10⁻⁷ M 9cRA (9c), or 10⁻⁶ M retinol (Rol). Electrophoretic mobility shift assays were performed with a radioactive DNA probe corresponding to nucleotides −51 through +84 of the HIV-1 LTR. (d) Cellular extracts from either untreated (−) or RA-treated (10⁻⁷ M; 4 days) (+) cells were incubated with radioactive, bromodeoxyuridine-substituted DNA probes corresponding to either nucleotides −51 through +84 or −51 through +12 of the HIV-1 promoter. Bound proteins were cross-linked to DNA by UV light and then separated on either 10% (left) or 6% (right) SDS polyacrylamide gels.