INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The molecular mechanisms underlying cytokine dysregulation following human retroviral infection are not well understood. Considerable data demonstrating aberrant cytokine production from both human T-cell leukemia virus type 1 (HTLV-1)- and human immunodeficiency virus type 1 (HIV-1)-infected cells have been obtained (37, 38, 67). Both viruses infect CD4⁺ T cells and monocytes/macrophages, cells which orchestrate the immune response primarily by the elaboration of cytokines. Therefore, elucidating the molecular mechanisms of cytokine dysregulation in HTLV-1- and HIV-1-infected cells is critical to understanding the mechanisms of pathogenesis of these viruses.

Methylation is an epigenetic mechanism for modulation of gene expression in mammalian cells (81, 83). Studies with knockout mice have demonstrated the absolute requirement for DNA methyltransferase (MTase), as homozygous mutant embryos die at mid-gestation (50). Faithful propagation of the methylation state (maintenance methylation) occurs directly after DNA replication. The process is mediated by an enzymatic methyl transfer reaction at cytosine located 5' to guanosine (CpG dinucleotide) residues in the unmethylated nascent DNA strand across from methylated CpG dinucleotides. Acquisition of DNA methylation at a previously unmethylated site cannot be accomplished by maintenance methylation and requires de novo methylation (48). To date, only one DNA (cytosine-5) MTase has been identified in mammalian cells (7, 45, 82). While this DNA MTase prefers a hemimethylated substrate, it shows both maintenance and de novo activity in vitro and in vivo (48).

A substantial body of work implicates a role for altered DNA methylation patterns and regulation in the pathogenesis of cancer (3, 42, 46). Changes in the pattern of DNA methylation are often seen in human tumors (2, 12, 22, 27, 29). One of these changes is the aberrant methylation of normally unmethylated CpG islands in gene promoter regions and an associated decrease in expression of tumor suppressor genes such as the von Hippel-Lindau (35), p16 (36, 54), and Rb (63) genes. While it is unclear whether the observed changes in DNA methylation play a direct role in oncogenesis or whether they are the result of the transformation process, the substantial correlative data and a recent study using a combination of genetics and pharmacology to decrease levels of DNA MTase in mice (46) strongly support a causal role for aberrant methylation in the pathogenesis of some cancers.

In other studies, it has been demonstrated that overexpression of murine DNA MTase is transforming for NIH 3T3 cells (79) and that levels of DNA MTase are increased in neoplastic cells (17, 44), with incremental increases at the different stages of colon carcinoma progression (5, 40). Some studies have found more modest (two- to fourfold) increases in MTase upregulation, which were correlated with increases in cellular proliferation, making it difficult to determine whether this increase was biologically significant or merely secondary to cell proliferation (47, 71). Nonetheless, recent studies illustrate that chronically increased cellular levels of DNA MTase can result in aberrant CpG island hypermethylation in simian virus 40-transformed human fibroblasts (74, 75). While it is unclear at present how such increased expression of DNA MTase results in increased tumorigenesis, it is known that methylated cytosine residues are susceptible to deamination, which changes methylcytosine to thymine, ultimately resulting in a permanent genetic alteration. 5-Methylcytosine is then a potential endogenous mutagen (68, 73). Alternatively, an increase in DNA methylation might enhance pathogenesis via an epigenetic mechanism by inhibiting the expression of tumor suppressor or cell cycle genes as discussed above.

While there is no published information concerning the role of DNA MTase in the pathogenesis of human retroviruses, it is known that the DNA of both endogenous and exogenous retroviruses can be highly methylated in the genome of the host (31, 52, 86) and that increased DNA methylation can occur elsewhere in the genome of infected cells (15, 41). In the case of the human retroviruses HIV-1 and HTLV-1, several studies have shown increased methylation of the viral long terminal repeats and throughout the viral genome, suggesting methylation as a mechanism of suppression of viral expression (4, 69) and latency (58, 59, 70).

The present study was initiated to determine whether infection by HIV-1 modulates expression of DNA MTase. Modulation of this enzyme could result in widespread aberrant methylation of genes, resulting in alteration of gene expression and ultimately contributing to the pathogenic effects of these viruses. Since it had been previously shown that methylation of the gamma interferon (IFN-) gene promoter could silence the expression of this gene in primary T cells and T-cell lines (20, 21, 24, 32, 53, 64, 66, 84, 85), we examined the effect of HIV infection on IFN- gene expression. We report here that acute infection of lymphoid cells by HIV-1 results in increased DNA MTase expression, an overall increase in methylation of DNA in infected cells, and the de novo methylation of a CpG dinucleotide in the IFN- gene promoter, resulting in the subsequent downregulation of expression of this cytokine. Antisense DNA MTase experiments demonstrated that this enzyme regulates the expression of IFN- in lymphoid cells. The use of IFN- as a target gene for aberrant DNA methylation provides support for a potential mechanism for some pathologic consequences of human retroviral infection involving altered methylation of genes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cell culture and separation of T-cell subsets. All cell lines, with the exception of NK 3.3, used in this study were maintained in RPMI 1640 supplemented with 10% fetal calf serum, penicillin (100 µg/ml), streptomycin (100 µg/ml), and L-glutamine (300 µg/ml) (complete RPMI). NK 3.3, whose growth is cytokine dependent, was grown in complete medium plus interleukin-2 (IL-2) (80). The JMO and CS-3 CD4⁺ cell lines were developed in our laboratory by HTLV-1 transformation of peripheral blood mononuclear cells (PBMCs). Clonal populations of JMO were prepared by standard limiting dilution methods. T lymphocytes were obtained from leukopaks isolated from healthy donors. Mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation followed by centrifugal elutriation to separate monocyte and lymphocyte populations as previously described (58). CD4⁺ and CD8⁺ T cells were obtained at >95% purity by incubating with specific monoclonal antibodies (MAbs) linked to magnetic beads and passed through a magnetic column (Milteny Biotec, Auburn, Calif.). Purity of populations was determined by flow cytometry analysis of purified populations. Cells were activated with phytohemagglutinin (PHA; 1 µg/ml) for 72 h and exposed to HIV-1 as previously described (58).

Cell cycle analysis of HIV-1-infected CD4⁺ T cells. Cell cycle analysis by flow cytometry was performed as follows. A total of 2 × 10⁶ cells were removed from infected and uninfected cultures at specified time points. Cells were washed twice with phosphate-buffered saline (PBS) and fixed in ice-cold ethanol for 1 h. Fixed cells were pelleted, resuspended in 0.5 ml of PBS containing propidium iodide (30 µg/ml) and RNase A (1 mg/ml; Sigma, St. Louis, Mo.), and analyzed for DNA content by flow cytometry. Cell cycle histograms were then analyzed with a multicycle computer program.

Plasmid constructs. Expression vectors for human DNA MTase were prepared as previously described (75). Clones were selected in the sense (HMT) and antisense (TMH) orientations. pCMV-Neo-Bam was used as a vector control in all stable transfections. For stable transfections (2 × 10⁶), cells were electroporated in complete RPMI at 250 V and 600 µF with 10 µg of plasmid. G418-resistant cells were isolated in selective medium (0.3 mg of G418 per ml; Life Technologies Inc., Gaithersburg, Md.). Clonal populations of G418-resistant cell lines were prepared by standard limiting dilution methods.

RPA. A 364-bp (bp 4774 to 5138) fragment of the 3' end of MTase subcloned into pBluescript (Stratagene, La Jolla, Calif.) was linearized with HindIII and in vitro transcribed, using a Promega (Madison, Wis.) RNA synthesis kit in the presence of [-³²P]UTP and T3 polymerase. The sense message (to detect a 280-bp antisense fragment) could be made by linearizing the same construct with DraI and using T7 polymerase in the transcription reaction. RNase protection assays (RPAs) were performed with an RPA II kit (Ambion) according to the manufacturer's instructions. A human 18S rRNA probe (116 bp) and human ptriactin RNA probe (225 bp), or ptricyclophilin (Ambion) in some experiments, were included in each reaction mixture as an internal loading standard. The multiprobe template human cytokine-1 (Pharmingen) was transcribed and used according to the RPA II (Ambion) protocol. Briefly, total RNA (20 µg) was hybridized at 50°C with the mixed radioactive probes. RNA was digested with RNase A and RNase T₁ at 37°C for 30 min, and protected fragments were precipitated and electrophoresed on a 6% sequencing gel. Gels were dried and scanned on a PhosphorImager (Molecular Dynamics, Palo Alto, Calif.), using Imagequant and Microsoft Excel software. Dried gels were also autoradiographed on XAR-5 film at 70°C.

Southern analysis. Five micrograms of DNA, isolated from uninfected and infected cells, was digested with a restriction enzyme (BamHI, SnaBI, HpaII, or MspI) overnight under appropriate conditions as specified by the manufacturer (Boehringer Mannheim or New England Biolabs). Restriction digests were ethanol precipitated analyzed by electrophoresis in 0.8% agarose gels as described elsewhere (9). Membranes were hybridized with ³²P-labeled full-length IFN- cDNA. The cDNA was labeled to a specific activity of 1 × 10⁸ to 2 × 10⁸ cpm/µg by random priming (Prime-it II; Stratagene) and transferred to a nylon membrane (Magna Graph; Micron Separations Inc., Westborough, Mass.). After hybridization, membranes were washed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate at room temperature for 20 min followed by a wash in 0.2× SSC-0.1% sodium dodecyl sulfate at 65°C for 20 min. Blots were then exposed to Kodak X-Omat X-ray film at 70°C overnight. Images were quantitated with a PhosphorImager using Imagequant (Molecular Dynamics). Blots were stripped and reprobed with mitochondrial DNA to control for complete digestion.

Derivation and maintenance of antigen-specific human CD4⁺ T-cell clones. Purified protein derivative (PPD)-, tetanus toxoid (TTx)-, and Dermatophagoides pteronyssinus antigen (DP)-specific as well as staphylococcal enterotoxin B (SEB)-reactive T-cell clones were generated according to previously described techniques (23). PPD and TTx were purchased from Connaught, Inc. (Swiftwater, Pa.), SEB was purchased from Sigma, and DP was purchased from Miles, Inc. (Spokane, Wash.). Briefly, PBMCs at a concentration of 5 × 10⁵ cells/ml in clone medium (EHAA [CLICK's] medium supplemented with L-glutamine, 2-mercaptoethanol, 2% human AB serum, 10% fetal calf serum, penicillin-streptomycin, nonessential amino acids, and sodium pyruvate) were stimulated with either PPD (1 µg/ml), TTx (10 µg/ml), DP (10 IAU/ml), or SEB (0.1 µg/ml) in 24-well flat-bottomed plates for 7 days. Many but not all of the T_H1 and T_H2 clones used in these studies were derived in cultures supplemented with the T-helper cell-selective cytokines IL-4 and IL-12. For the generation of T_H1 clones, these cultures were supplemented with recombinant human IFN- (rhIFN-; 10 U/ml), rhIL-12 (50 pg/ml), and anti-IL-4 MAb (10 µg/ml) over the culture period. For T_H2 clones, bulk cultures were supplemented with rhIL-4 (200 U/ml) and anti-IFN- MAb (10 µg/ml). Forty-eight hours after the initiation of the cultures, rhIL-2 (10 U/ml) was added to each well. After 7 days of incubation, the cultures were harvested, extensively washed, replated in fresh clone medium supplemented with additional IL-2 (10 U/ml), and incubated for an additional 7 to 10 days. Viable T cells were then plated in limiting dilution cultures (0.5 cells/well) in 16 flat-bottomed 96-well plates containing 2 × 10⁵ irradiated (1,200 rads) syngeneic PBMC feeder cells, specific antigen, and IL-2 (10 U/ml) in a final volume of 200 µl. The growing cultures were examined daily and supplemented with the T_H1- and T_H2-selecting cytokines (as described above) and at 10-day intervals with feeder cells and IL-2. Individual clones were isolated, characterized for lymphokine production by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR (RT-PCR) analysis and for the ability to respond to specific antigen in combination with syngeneic irradiated (1,200 rads) feeder cells. In maintaining T-helper clones, every 14 to 21 days, cells were restimulated with specific antigen in the presence of autologous PBMCs treated with mitomycin C at 25 µg/ml to prevent outgrowth of feeder cells and IL-2 (20 U/ml). After 96 h of antigenic stimulation, cells were subjected to two successive Ficoll-Hypaque centrifugations to remove dead cells. All clones were tested for their cytokine profile by ELISA and RT-PCR after stimulation with phorbol myristate acetate and monoclonal anti-CD3 as well as with antigen and antigen-presenting cells to phenotype each clone.

HIV infection of T cells and T-cell clones. PHA-activated T cells, CD4⁺ T cells 3 days after activation, and antigen-stimulated clones (10⁶ cells) 4 to 7 days postactivation (>95% viable by trypan blue exclusion) were inoculated with cell-free viral isolates at a multiplicity of infection (MOI) of 1 for 2 h before complete aspiration of medium, washing with PBS, and addition of fresh growth medium containing IL-2 (10 U/ml). Cells were aliquoted at 10⁶ cells/ml in 24-well plates or tissue culture flasks. Cell lines were infected with cloned isolates or purified concentrated isolates at an MOI of 1. Three laboratory strains of HIV (BP-1 [59], ADA [26], and MN) as well as the defined clones NL43 and HXB-2 (stocks purified 1,000-fold; kindly provided by the AIDS Vaccine Program, Frederick, Md.) were used.

Cytokine mRNA expression of T-helper clones. RT-PCR was also used to pedigree cell clones for cytokine expression. Primer pairs for IFN- and IL-4 (Clontech) and HIV SK38/39 were used to amplify cDNA prepared by Superscript II reverse transcription of 5 µg of RNA (GIBCO-BRL) according to the manufacturer's instructions. Amplification was carried out by using [-³²P]dCTP as described previously (16); products were electrophoresed on 8% polyacrylamide gels and then subjected to autoradiography.

Quantitation of cytokine and p24 production by T-cell clones. Quantitative determinations for each of the lymphokines from the 48-h supernatant of antigen-stimulated T-cell clones were determined by human IL-2, IL-4, IL-5, IL-10, and IFN- ELISAs (Quantikine; R&D Systems) as instructed by the manufacturer. The results are expressed in either nanograms/milliliter or units/milliliter based on a standard curve using recombinant cytokine within the ELISAs. Cytokine analyses after HIV infection were performed with cell-free supernatant, and the results were quantitated by ELISA for IL-4, IL-5 (R&D Systems), and IFN- (Medigenix) with sensitivities of 3 pg/ml, 1 pg/ml, and 1 IU/ml, respectively. Viral p24 antigen was determined by ELISA (Cellular Products, Buffalo, N.Y.) with a sensitivity of 10 pg/ml.

PCR analysis of IFN- promoter methylation status. Primer pairs were chosen such that they flanked the methyl-sensitive SnaBI site at 55 of the IFN promoter. The upstream (US) SnaBI sense primer 31-48 with the antisense (AS) primer 870-890 yielded a product of ~850 bp. An internal control primer, the downstream sense primer (DS) 319-342 with the AS 870-890 primer yielded a product of ~470 bp and was used as an internal control. DNA aliquots of 200 ng to 1 µg were digested with BamHI and SnaBI overnight to ensure complete digestion; 20 ng of DNA was then amplified in a 50-µl PCR mixture containing 0.25 µl of [³²P]dCTP as described above. Cycling conditions were 1 cycle of 94°C for 45 s, 60°C for 60 s, and 72°C for 45 s and 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by extension for 7 min at 72°C. PCR products were electrophoresed on 8% polyacrylamide gels, dried, autoradiographed, and quantitated on a PhosphorImager using Imagequant (Molecular Dynamics). Alternatively, 100 ng of DNA was amplified in a 50-µl reaction mixture with unlabeled deoxynucleoside triphosphates, and products were visualized by ethidium bromide staining following polyacrylamide gel electrophoresis. Quantitation was performed by densitometric analysis of gels photographed with Polaroid type 55 Pos/Neg film.

DNA methyltransferase enzymatic activity assay. Cell lysates were prepared as previously described (40). Briefly, cells were lysed by sonication at 0°C in 5 volumes of 20 mM Tris-HCl (pH 7.4)-0.4 M NaCl-25% glycerol-5 mM EDTA-0.1% Nonidet P-40-1 mM dithiothreitol (DTT)-0.2 mM phenylmethylsulfonyl fluoride-100 µg of aprotinin per ml. After sonication, 1 volume of a 50% Chelex-100 resin was added, and the mixture was incubated for 10 min to remove nucleic acids from the lysate. The clarified lysate (~200 mg of protein in 20 µl) was added to 200 µl of a solution containing 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 25% glycerol, 5 µCi of S-adenosyl-L-[methyl-³H]methionine (12 Ci/mmol; New England Nuclear), 4 µg of poly(dI-dC) · poly(dI-dC), 1 mM DTT, and bovine serum albumin (200 µg/ml). The assay mixture was then incubated at 37°C for 2 h and extracted twice with phenol-chloroform. The aqueous phase was made 0.1 M in NaOH and incubated at 50°C for 2 h. The solution was neutralized with HCl, and the radioactivity incorporated into DNA was measured by scintillation counting following trichloroacetic acid precipitation.

DNA methylation level. A modified methyl accepting assay (79) was used to determine the methylation status of DNA isolated from uninfected and infected primary cell culture cultures, chronically infected cell lines, and the stably transfected transformed cell line JMO. DNA (200 ng) was incubated with 4 U of M · SssI CpG methylase (New England Biolabs) in the presence of 1.5 µM S-adenosyl-L-[methyl-³H]methionine (60 to 85 Ci/mmol; Amersham product no. TRK 581) and 1.5 µM nonradioactive S-adenosylmethionine (New England Biolabs). The reaction mixtures (20 µl) were incubated at 37°C for 4 h in a buffer containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl₂, and 1 mM DTT. The reactions were stopped by the addition of 5 µl of 2.5 mM nonradioactive S-adenosylmethionine and spotted on GF/C 2.4-cm² Whatman filter discs, which were air dried for 15 min and washed with 6 ml of 5% (wt/vol) trichloroacetic acid-70% (vol/vol) ethanol. Discs were counted in Econofluor in a Beckman liquid scintillation counter. Control reactions without DNA or enzyme added were included as background, and counts in the samples never exceeded 5% of those in the test samples. All samples were tested in triplicate, and values were obtained as disintegrations/minute per nanogram of DNA.

ELISPOT for IFN- expression. The frequency of specific IFN--producing cells was determined by ELISA spot assay (ELISPOT). Briefly 96-well nitrocellulose-bottomed plates (Millititer HA; Millipore Inc., Bedford, Mass.) were coated with specific mouse anti-human IFN- antibody, clone 1-D1k (Chromigenix, Montal, Sweden) by overnight incubation at 4°C. Cells to be analyzed were washed twice in appropriate incubation media. A known number of cells was then plated to the top row of the previously IFN- MAb-coated plate, serially twofold diluted down the plate, and incubated at 37°C for 16 h. Cells were then washed off, and IFN--secreting cells were detected with a biotinylated second IFN--specific antibody, clone 7-B6 (Chromigenix). Plates were next incubated with horseradish peroxidase-conjugated avidin and developed with TruBlu peroxidase substrate (Kirkegaard & Perry, Gaithersburg, Md.) to produce an insoluble precipitate that formed distinct spots that could be easily counted by light microscopy.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

HIV-1 infection of lymphoid cells increases cellular capacity to methylate DNA by increasing DNA MTase expression. To determine whether there is a relationship between retroviral infection and the overall cellular capacity to methylate DNA, we first examined the time-dependent effect of acute HIV-1 infection of primary T cells and T-cell subsets on DNA MTase mRNA expression. As shown by RPA (Fig. 1A), a significant increase in DNA MTase mRNA was observed in PHA-activated, HIV-1-infected T cells compared to their PHA-activated, uninfected counterparts on days 4, 7, and 10 postinfection. Similarly, when CD4⁺ T cells were isolated, activated with PHA, and infected with HIV-1, levels of DNA MTase mRNA expression were higher in infected cells than in uninfected cells throughout a similar 10-day incubation period (Fig. 1B). This increase in DNA MTase RNA was seen with several T-cell-tropic HIV-1 isolates (BP-1, IIIB, and a clinical isolate) (data not shown). Similar results were obtained when CD4⁺ Hut 78 cells were infected with the molecularly cloned proviruses NL43 and HXB-2 (Table 1). Increases of 3- to 20-fold in MTase RNA could be seen as early as 2 days postinfection in both primary T cells and the T-cell line Hut 78. 

View larger version (46K): [in this window] [in a new window]   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.

View larger version (50K): [in this window] [in a new window]   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%.

View larger version (30K): [in this window] [in a new window]   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).

Increased genomic methylation in primary cells and cell lines following acute infection with HIV-1. Having established that HIV-1 infection can increase the cellular capacity to methylate DNA, we examined whether this was reflected in an overall increase in genomic methylation of the infected cell population. This was assessed two ways. First, genomic DNA was digested with methyl-insensitive and -sensitive restriction enzymes (MspI and HpaII) followed by Southern blot analysis with an Alu-specific probe. Increased overall methylation was demonstrated after HIV infection, as mainly high-molecular-weight DNA was seen with the methyl-sensitive HpaII while the methyl-insensitive MspI gave low- as well as high-molecular-weight bands (data not shown). Second, a modified methyl accepting assay (79) was used to more quantitatively assess the methylation status of DNA isolated from uninfected and infected primary cell cultures as well as following acute infection of human cell lines. As shown in Table 3, an increase in overall genomic methylation was seen as early as 2 days after acute HIV-1 infection of both primary PHA-activated CD4⁺ T cells and the T-cell line Hut 78 compared to uninfected control cultures. The lack of cell death at day 2 postinfection indicates that HIV-1 infection does not select for a subpopulation of cells expressing higher levels of DNA MTase. By day 7, methylation levels of DNA in the HIV-1-infected cultures were as high as 152% of the level of uninfected controls. Daily cell proliferation measurements postinfection by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of infected cultures were similar to or slightly less than those for the uninfected cultures (data not shown).

De novo methylation of the IFN- promoter occurs in T_H1 cells following acute infection with HIV-1. A recent study has shown that the cellular DNA MTase can directly affect the de novo methylation of CpG islands if enzyme levels are increased ninefold (75). As we consistently saw high levels of DNA MTase activity in T_H1 clones following acute infection with HIV-1, we next sought to identify genomic targets of this increased methylation, particularly sites which may become de novo methylated. In this regard, of the many genes that would be potential candidates for aberrant methylation during HIV infection, we chose IFN-, since previous studies have demonstrated that the IFN- promoter contains a methyl-sensitive endonuclease SnaBI site at position 52 of the transcription start site (22, 65, 84) in a region of the promoter critical for promoter activity (22, 65). Further, the methylation status of the promoter at this site correlates with IFN- gene expression, as human and murine T_H1 cells, large granular lymphocyte cells, and peripheral blood memory T cells, which express IFN-, show hypomethylation of the IFN- core promoter at this site (57, 84). Cells that do not express IFN-, such as T_H2 cells and naive T cells, are hypermethylated at this site (22, 57, 84). Given the important role of IFN- both in the immune response to viral infections and in the development of T_H1 cells (60), and for the reasons outlined above, we hypothesized that the IFN- gene represented a potential target of the increased DNA MTase activity demonstrated in HIV-1-infected T cells.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Southern analysis of the methylation status of the IFN- 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.

View larger version (28K): [in this window] [in a new window]   FIG. 5.   PCR analysis of IFN- 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- 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.

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Correlation of HIV-1 infection and IFN- mRNA expression with methylation state of the IFN- promoter in TH1 clones. RNA or DNA was isolated at day 7 following infection. (A) IFN- 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- mRNA in the same TH1 clone used for panel A. Film was overexposed to demonstrate lack of IFN- mRNA expression 7 days following HIV-1 infection.

View this table: [in this window] [in a new window]   TABLE 4.   HIV-1 infection of human TH1 clones downregulates IFN- expression

Antisense constructs to DNA MTase prevent IFN- gene methylation and increase IFN- production. All of the above data predict that in HIV-1- and HTLV-1-infected lymphocytes, decreasing DNA MTase activity and the cellular capacity to methylate DNA will increase the expression of the IFN- gene. To test this directly, we used an antisense gene insertion approach to modulate DNA MTase expression. We achieved stable expression of a DNA MTase antisense construct in a lymphoid cell line, designated JMO (CD3 CD4⁺ CD2 CD8 CD19 CD14 CD25⁺ CD16 CD56⁺), developed in one of our laboratories. The parental JMO cell line and all single-cell clones of this cell line were shown to constitutively express detectable but variable levels of DNA MTase and IFN- as measured by RPA and ELISPOT, respectively (data not shown). To verify that HIV-1-induced DNA MTase expression could modulate IFN- expression in this cell line, JMO clone D8, whose basal MTase expression was lower (compare lanes 1 in Fig. 7A and 8A) and IFN- expression was higher (Table 5) than that of the parental JMO cell line, was infected, and total RNA was prepared 7 days postinfection. The RNA was analyzed by RPA as described in Materials and Methods. As shown in Fig. 7A, HIV-1 infection upregulated DNA MTase in JMO. Figure 7B shows a concomitant downregulation of IFN- mRNA in a multiprobe RPA to detect human cytokines (Pharmingen). Interestingly, the expression of IL-15 and IL-9 remained unchanged (Fig. 7B and C), demonstrating that there is not a general downmodulation of cytokine expression in the HIV-1-infected cells.

View larger version (27K): [in this window] [in a new window]   FIG. 7.   HIV-1 infection regulates MTase and IFN- 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- 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.

View larger version (36K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   TABLE 5.   Increased IFN- production in cells transfected with an antisense MTase vector

View larger version (45K): [in this window] [in a new window]   FIG. 9.   DNA MTase expression regulates IFN- 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- 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- 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.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

HTLV-1 and HIV-1 cause an overlapping set of pathogenic events including tumor development, immunodeficiency, and neurological symptoms (37, 49). Both human retroviruses preferentially infect CD4⁺ T lymphocytes, monocytes, and macrophages. It is the interplay of these cell types that orchestrates an immune response, primarily through the elaboration of cytokines. In the past decade, many reports have documented dysregulation of cytokines in vivo and in vitro following infection with these viruses (37, 38, 67). However, little is known about the molecular mechanism(s) involved in this cytokine dysregulation. In this report, we have shown that infection by HTLV-1 and HIV-1 can upregulate DNA MTase expression and increase the cellular capacity to methylate genes. Increased DNA MTase expression is seen not only in acutely infected cell lines of T-cell, NK cell, and monocyte/macrophage lineages but also following acute HIV infection of primary T cells and monocytes. We have shown herein that this increase in the cellular capacity to methylate genes not only results in an overall increase in DNA methylation in the infected cells but also has the capacity for de novo methylation. Among many possible candidate genes, we have identified one, the IFN- gene, and demonstrated de novo methylation at a site which had previously been shown to be critical in the transcriptional regulation of this gene.

Young et al. have reported (84, 85) that methylation of a CpG dinucleotide at 55 of the transcriptional start site of the IFN- could negatively regulate the interaction of nuclear DNA binding proteins with this site. They further demonstrated that this site is differentially methylated in the T_H1 and T_H2 helper T-cell subsets. T_H1 cells, which produce IFN-, are hypomethylated at this site, while T_H2 cells, which do not produce IFN-, are hypermethylated at this site. Previous studies showed that 5-azacytidine, a hypomethylating agent, stimulates IFN- production in cytotoxic T cells as well as in T_H2 cells. These studies also demonstrated the specificity of this methylation, as expression other cytokines is not altered in these systems (21, 32, 84). One of the mechanisms by which DNA methylation has been shown to inhibit transcription is by inhibiting the recognition of the sequence motif by a transcription factor (39). Young et al. (84, 85) have also shown decreased levels of specific DNA binding protein complexes in T_H1 cell nuclear extracts following in vitro methylation of this SnaBI site. It is of note that this site is not part of a CpG island, as there are only five CpG dinucleotides in the first 780 bp of the human IFN- promoter (Fig. 5A). Furthermore, none of the other CpG sites have been demonstrated to directly interact with DNA binding proteins. Here we detect marked increases in IFN- promoter methylation in lymphocytes following infection by HIV-1 or HTLV-1. Furthermore, we show that acute HIV-1 infection of T_H1 clones in vitro results in increased DNA MTase activity, methylation of the SnaBI site in the promoter of the IFN- gene and subsequent downregulation of IFN- mRNA, and protein production from these infected cells. These results demonstrate a strong correlation during viral infection between increased DNA MTase levels, methylation of the IFN- promoter, and decreased IFN- production. Using an MTase antisense strategy, we observed a direct relationship between DNA MTase and IFN- gene expression through regulation of the methylation of the IFN- promoter.

The mechanism of this upregulation of DNA MTase expression during retroviral infection is not clear. Since DNA MTase activity is tightly linked to cellular proliferation, and some argue that in carcinogenesis, MTase upregulation is secondary to cell growth (47), it is possible that the upregulation of MTase activity by HIV-1 is secondary due to increases in cell proliferation. However, no difference in cell number, thymidine incorporation, or cell cycle status was seen between infected and uninfected primary CD4⁺ T cells. Moreover, infection of Hut 78, a continually proliferating T-cell line, with NL43 and HXB-2 resulted in rates of proliferation lower in the infected cells than in the mock-infected cells. Further, in 18-day monocyte cultures under conditions where labeling indices for tritiated thymidine were reported to be 1 positive cell per 1,000 whether or not the cells were HIV infected (77), high DNA MTase RNA expression is seen only in infected cultures. Whether or not proliferation is needed for initiation of HIV-1 infection, it is clear from the results presented here that HIV-1 infection maintains increased levels of DNA MTase expression in the absence of proliferation. On the other hand, since high levels of DNA MTase can be toxic to cells (75, 78), this MTase upregulation could be a nonspecific response to MTase-induced apoptotic signals. However, this is unlikely since approximately 20 to 30% of the cultured uninfected T cells were apoptotic by 10 days postinfection, due to a lack of IL-2, and no DNA MTase activity was detected in these cultures. Furthermore, the rapid increase in DNA MTase expression, overall genomic methylation, and decrease in IFN- production at the single-cell level by day 2 postinfection, in the absence of detectable cell death, indicates that the selection of a rare cell with these characteristics is not occurring. In addition, using a one-step assay where zidovudine inhibits viral replication, Fan et al. (20) also showed downregulation of IFN- mRNA in HIV-infected Hut 78 cells. Zidovudine blocked this downregulation, suggesting that the downregulation was a direct effect of viral replication.

These results provide a potential mechanism for dysregulation of expression of many genes following human retroviral infection. The overall increase in genomic methylation seen after HIV infection could have a toxic effect on the cells. In fact, a significant proportion of the primary cell cultures undergo HIV-1-induced apoptosis whether infected in vivo or in vitro (57). It is not known if the high level of MTase activity plays a role in this cell death; however, it should be noted that increases in MTase expression above a certain threshold are toxic (75). In addition, our finding that by increasing the cellular capacity to methylate genes, there is one gene that HIV-1 can downregulate, the IFN- gene, a key element in the host defense against viral infection, is of particular interest. The role of IFN- in differentiation of precursors of T_H1 cells, downregulation of the production of T_H2 cells, activation of monocytes to resist foreign invaders, and prevention of cellular spread of viruses has been well documented (60). Compromising any one of these functions could have an effect on some of the pathological consequences of HIV infection. Shearer and colleagues (13, 72) have hypothesized that there is an induction of a T_H2 bias during HIV-1 infection and progression to AIDS. Our present data support this hypothesis in two ways. First, since IFN- expression is a requirement for designating a cell T_H1, methylation of the IFN- promoter could lead to a low estimate of the number of T_H1 cells in a population. Second, decreased IFN- expression could result in a decrease in production of T_H1 cells (8) as well as a decrease in monocyte functions such as IL-12 production, which is observed in AIDS patients (11, 25). The presence of IFN- is needed for the long-term production of IL-12 in monocytes (51). Previous studies have suggested that the progression to AIDS is, at least in part, dependent on the ability to generate a T_H1-like immune response (72). Specifically, aberrant methylation of the IFN- promoter could play a role in the gradual loss of type 1 response seen in AIDS patients.

Though beyond the scope of this study, these results indicate the need to define which, if any, of these potential pathogenic effects resulting from the aberrant methylation of the IFN- gene or any other gene are important in AIDS. The role of IFN- in the development of AIDS is not clear. Consistent with a decrease in T_H1 function, several studies show a decrease in IFN- production (23, 38, 61, 87). In contrast, increased IFN- production has been reported (28, 62, 76). Since IFN- is produced by the CD4⁺ target cells of HIV-1 as well as cells reactive to HIV-1 infection (e.g., large granular lymphocyte and CD8⁺ T cells), these discrepancies in IFN- production could result from the fact that most studies have quantitated systemic IFN- in vivo or after short stimulation in vitro of bulk PBMC cultures from AIDS patients, potentially masking differential effects on target cells. Indeed, recent studies have shown that most IFN- is produced by CD8⁺ cells either in the periphery (28) or in the lymph nodes (19), where most viral replication occurs during the asymptomatic phase of AIDS (18). Single-cell analysis of IFN- production in T cells from HIV-1-infected individuals showed decreased numbers of CD4⁺ IFN--producing cells but preserved numbers of IL-4-producing cells (55, 56). No effect was seen on the numbers of CD8⁺-producing cells. The effects of this decrease in CD4⁺ IFN--producing cells on the cellular immune response and control of viral spread, particularly during acute infection, need to be determined. In this regard, recent evidence suggests that controlling viral load and spread is critical in determining the extent and progression of AIDS (6, 10, 43). If this variable course of lentiviral infection and long-term outcome is related to controlling viral spread, the number of CD4⁺ IFN--producing cells present during HIV-1 infection could be a determinant of this control. We are now studying this question in a simian immunodeficiency virus primate model.

Our current findings may have implications for virus-induced tumorigenesis. Previous studies have shown that the promoter region of the calcitonin gene becomes densely methylated following cell infection with multiple tumorigenic viruses, including simian virus 40, Epstein-Barr virus, and HTLV-1 (15). Our results suggest that the underlying mechanism for this change could involve the early increases that we now define for DNA MTase activity following HIV and HTLV-1 infection of cells. While this study has focused on the de novo methylation of only one gene, the increases in overall genomic methylation demonstrated herein suggest that many genes may be altered by this mechanism during viral infection. The fact that multiple tumor suppressor genes are now known to be inactivated in association with aberrant promoter region methylation (34-36, 54) suggests that altered methylation of genes could be studied as a candidate early step in the tumorigenic activity of a range of viruses.