Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production

Abstract

The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-gamma), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-gamma, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-gamma gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-gamma gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-gamma gene, and increased IFN-gamma production, demonstrating a direct link between methyltransferase and IFN-gamma gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-gamma gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

Figures

FIG. 1

Expression of DNA MTase in HIV-infected PHA-activated T cells and purified T-cell subsets detected by RPA as described in Materials and Methods. Data shown are representative of four normal donors and five TH1 cell clones. Each experiment was done with a different healthy donor. (A) Elutriated PHA-activated lymphocytes; (B) purified PHA-activated CD4+ T cells; (C) TTx-specific TH1 clone H1.15. Days after infection are as indicated.

FIG. 2

Schematic depiction of DNA MTase activity in primary T cells with time following HIV-1 infection. DNA MTase activity was determined in infected cell lines maintained in log-phase growth and primary T cells following HIV-1 infection using S-adenosyl-l-[methyl-3H]methionine as the methyl donor and poly(dI-dC) · poly(dI-dC), which acts similarly to hemimethylated DNA, as the substrate as described in Materials and Methods. Primary CD4+ T cells from the same donor were activated with PHA for 3 days, and one set was infected with HIV-1 (day 1 on the graph). Parallel cultures of infected and uninfected cells from the same donor were harvested at daily intervals following infection. Relative values are given as the fold increase above that seen in day 3 activated T cells, which is set to equal zero. Data shown are the average of three separate experiments with a standard error of the mean of <20%.

FIG. 3

Expression of DNA MTase in 14-day MDM detected by RPA as described in Materials and Methods. Data shown indicate purified MDM (>94) isolated from two different healthy donors. Infection with HIV-1 monocytotropic strain Bal was performed 3 days following plastic adherence of monocytes. Positive infection was determined by p24gag ELISA on cell supernatants. Total RNA was isolated at day 14 postinfection (18 days of culture).

FIG. 4

Southern analysis of the methylation status of the IFN-γ promoter at the SnaBI site in cell lines with or without HTLV-1 and HIV-1 infection. Digestions and analysis were performed as described in Materials and Methods. B, BamHI digest alone; B/S, BamHI and SnaBI digest of DNA. Group 1, HTLV-1-infected NK 3.3; group 2, uninfected NK 3.3; group 3, THP-1; group 4, HIV-ADA-infected THP-1.

FIG. 5

PCR analysis of IFN-γ promoter methylation status in T-cell clones and lymphoid cell lines. (A) (Top) Schematic showing the PCR primer locations (US, DS, and AS), the HpaII sites, the SnaBI site, and the CpG promoter sites in the IFN-γ gene. Solid boxes represent exons, and open boxes represent introns. (Bottom) When DNA was digested with SnaBI, PCR with the US primer pair plus the AS primer, which flank the SnaBI site, yields a product if the SnaBI site is methylated (HTLV-1-infected NK cells) but no product if the site is unmethylated (NK cells). As a DNA loading control, PCR was performed with the DS-AS primer pair, which does not flank the SnaBI site and should amplify independent of methylation status. Identical PCR products are generated from both the infected and the uninfected cells. (B) PCR with the US-AS primers in DNA, from various cells, digested (+) and not digested (−) with SnaBI. Group 1, uninfected human TH1 cell clone; group 2, uninfected human TH2 cell clone; group 3, uninfected NK 3.3 (NK cell line); group 4, NK 3.3 14 days after HTLV-1 infection; group 5, uninfected CS-3 (T cells); group 6, CS-3 7 days after HIV-1 infection.

FIG. 6

Correlation of HIV-1 infection and IFN-γ mRNA expression with methylation state of the IFN-γ promoter in TH1 clones. RNA or DNA was isolated at day 7 following infection. (A) IFN-γ promoter methylation status of the SnaBI site in SnaBI-digested DNA from an HIV-infected and uninfected TH1 clone as detected by the PCR procedure outlined for Fig. 4. (B) RT-PCR analysis of IFN-γ mRNA in the same TH1 clone used for panel A. Film was overexposed to demonstrate lack of IFN-γ mRNA expression 7 days following HIV-1 infection.

FIG. 7

HIV-1 infection regulates MTase and IFN-γ expression in a lymphoid cell line. The T-cell line JMO clone D8 was infected with HIV-1. At 7 days postinfection, MTase and IFN-γ expression was measured, using 5 μg of total RNA in each RPA as described in Materials and Methods. (A) MTase RPA. Lane 1, uninfected JMO clone D8; lane 2, HIV-infected JMO clone D8. (B) Cytokine RPA. Lane 1, uninfected JMO clone D8; lane 2, HIV-infected JMO clone D8. Probe, human cytokine-1 multiprobe (Pharmingen). (C) PhosphorImager (Molecular Dynamics) quantitation of results in panels A and B, normalized to the actin and L32 controls, respectively.

FIG. 8

DNA MTase expression in stably transfected lymphoid cells. As described in Materials and Methods, the parental cell line JMO was stably transfected with an antisense DNA MTase expression vector to generate JMO-TMH cells and with the vector alone to derive the JMO-neo control cells. RPA of MTase sense and antisense expression was determined in the lymphoid cells. (A) Probe alone. Lane 1, JMO-neo; lane 2, JMO-TMH. (B) Probe alone. Lanes 1 and 2, clones of JMO-neo; lanes 3 and 4 clones of JMO-TMH.

FIG. 9

DNA MTase expression regulates IFN-γ expression in lymphoid cells. As described in Materials and Methods, the parental cell line JMO was stably transfected with an expression vector containing the full-length DNA MTase cDNA, in the antisense orientation, to generate JMO-TMH, with the vector alone to generate JMO-neo control, and with the sense-orientation DNA MTase cDNA to derive JMO-HMT as an additional control. (A) RT-PCR analysis of the expression of IFN-γ mRNA in these lymphoid cell lines. Lane 1, JMO-neo; lane 2, JMO-HMT (sense); lane 3, JMO-TMH (antisense). GAPDH mRNA is also shown as internal loading control for semiquantitative PCR. (B) Southern analysis of the methylation status of the IFN-γ region SnaBI site and first intronic region HpaII site. BamHI, BamHI-SnaBI, and BamHI-HpaII digests were performed as described in the text. Lanes 1, 4, and 7, JMO-neo control; lanes 2, 5, and 8, JMO-TMH, DNA MTase cDNA in the antisense orientation; lanes 3, 6, and 9, JMO-HMT, DNA MTase cDNA in the sense orientation.