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Molecular and Cellular Biology, September 1998, p. 5166-5177, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of
the Gamma Interferon (IFN-
) Promoter and Subsequent Downregulation
of IFN-
Production
Judy A.
Mikovits,1,*
Howard A.
Young,2
Paula
Vertino,3
Jean-Pierre J.
Issa,3
Paula M.
Pitha,3
Susan
Turcoski-Corrales,4
Dennis D.
Taub,5
Cari L.
Petrow,1
Stephen B.
Baylin,3 and
Francis
W.
Ruscetti6
Intramural Research Support Program, SAIC
Frederick,1 and
Laboratory of
Experimental Immunology2 and
Laboratory of Leukocyte Biology,6
Division of Basic Sciences, National Cancer Institute-Frederick Cancer
Research and Development Center, Frederick Maryland 21702-1201;
Oncology Center, The Johns Hopkins Medical Institutions,
Baltimore, Maryland 212313;
CBER,
Rockville, Maryland 208924; and
Laboratory of Immunology, National Institute of Aging,
Baltimore, Maryland 212245
Received 3 March 1998/Returned for modification 28 April
1998/Accepted 23 June 1998
 |
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-
), 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-
, 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-
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-
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-
gene, and increased IFN-
production,
demonstrating a direct link between methyltransferase and IFN-
gene
expression. The ability of increased DNA methyltransferase activity to
downregulate the expression of genes like the IFN-
gene may be one
of the mechanisms for dysfunction of T cells in HIV-1-infected
individuals.
 |
INTRODUCTION |
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 |
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
TH1 and TH2 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 TH1 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 TH2 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 TH1- and TH2-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 |
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.

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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.
|
|
As CD4+ helper T cells can be further subdivided into
subsets based on their cytokine profiles (human TH1 cells
express IFN-
but not IL-4 and IL-5, while TH2 cells
express IL-4 and IL-5 but not IFN-
), we next examined the effect of
HIV-1 on DNA MTase mRNA expression in antigen-specific human
TH1 and TH2 clones following infection by
HIV-1. In five antigen-specific TH1 clones and three antigen-specific TH2 clones, DNA MTase mRNA expression was
8- to 20-fold higher in the infected TH1 and
TH2 clones than in the uninfected controls. A
representative experiment is shown in Fig. 1C. DNA MTase mRNA
expression was greatly upregulated at both day 5 and day 10 following
HIV-1 infection in a TH1 clone compared to the expression
of DNA MTase mRNA in the uninfected clone (Fig. 1C). These findings
show that acute infection of CD4+ T cells, including both
TH1 and TH2 T-cell subsets, with HIV-1 markedly upregulates DNA MTase mRNA expression.
We next examined whether DNA MTase mRNA levels following acute HIV-1
infection are associated with increased cellular DNA
MTase activity. As
expected, DNA MTase activity was upregulated
in uninfected cells
following PHA stimulation of T-cell proliferation.
Limiting amounts of
exogenous IL-2 (10 U/ml) were added to CD4
+ T cells after
HIV-1 infection, which occurred 3 days after PHA
activation. As a
result, detectable DNA MTase activity in uninfected
cells declined as
proliferation declined, and no MTase activity
was found at later time
points (Fig.
2). In contrast, by day 4
following infection, DNA MTase activity in HIV-1-infected cells
further
increased above the level in uninfected control cells.
By day 5, HIV-1-infected cells displayed peak levels of HIV-1
p24 production
(data not shown) and levels of DNA MTase activity
threefold higher than
in uninfected cells. Under these culture
conditions, the T cells cease
proliferating between 7 and 10 days
postinfection (10 to 13 days after
PHA activation), as measured
by cell counts and tritiated thymidine
incorporation. In fact,
a significant proportion of the primary cell
culture undergoes
HIV-1-induced apoptosis at day 7 and later time
points following
infection. It is not known if high levels of MTase
activity play
a role in this cell death. However, it was previously
noted that
increases in MTase expression above a certain threshold are
toxic
(
75). Despite the lack of cell proliferation, a
substantial
level of MTase activity was present in the infected cells
at day
10, while no increased MTase activity was detectable in the
uninfected
CD4
+ T-cell control (Fig.
2).

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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%.
|
|
Since DNA MTase activity is tightly linked to cell proliferation, it is
important to determine whether this upregulation of
DNA MTase activity
by HIV-1 infection is secondary to increased
cellular proliferation, as
has been suggested for tumorigenesis
models (
47,
71). There
is no compelling evidence that HIV-1
infection stimulates T-cell
proliferation. Indeed, several studies
show that it induces apoptosis
(
30,
56) and/or, through the
vpr gene encoded by
HIV-1, induces cell cycle arrest in the G
2 phase of the
cell cycle (
1,
33). Also, infection of Hut 78,
a
continuously proliferating T-cell line, with NL43 and HXB-2
(Table
1)
resulted in rates of proliferation 10 to 15% lower
in the infected
cells than in the mock-infected cells. Nonetheless,
to further rule out
that the differences in DNA MTase expression
seen were due to the
effects of the virus on cell proliferation,
cell cycle analysis by
propidium iodide staining and flow cytometry
was performed on both the
uninfected and infected cultures throughout
the time course of the
infection. No significant differences were
seen in the cell cycle
progression between the HIV-1-infected
and uninfected CD4
+
T cells up to 10 days postinfection (Table
2).
In addition, because the HIV-1 accessory gene
vpr is also
required for efficient replication of the virus in primary macrophages
associated with its function in facilitating nuclear localization
in
these nondividing cells (
14), we examined MTase mRNA levels
in primary nondividing monocyte-derived macrophages (MDM) from
normal
donors. In MDM from two different donors expressing similar
levels of
HIV-1 p24 at 14 days after infection with the HIV-1
Bal isolate, the
MTase mRNA levels were three- and fivefold higher
in the infected
culture than in the uninfected matched culture
(Fig.
3). At that time, these cultures did not
incorporate tritiated
thymidine (data not shown). This finding provides
further evidence
that viral infection leads to upregulation of the
MTase at the
cellular level. Thus, it is likely that HIV-1 infection
maintains
increased levels of DNA MTase expression independent of
proliferation.
The mechanism of how HIV-1 infection maintains this
upregulation
of MTase activity is not clear. Expression of single gene
products
of HIV, including Nef, Tat, and Rev, using transient and
stable
transfection of human T-cell lines has not shown any clear
ability
to upregulate MTase mRNA expression (data not shown).

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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).
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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
TH1 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 TH1 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 TH1 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 TH2 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 TH1 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.
We first examined by Southern analysis the methylation status of the
IFN-

promoter in cell lines chronically infected with
HIV-1. As a
control for these studies, we also used an NK cell
line which had been
chronically infected by HTLV-1, as it had
previously been shown that
the IFN-

promoter in this uninfected
cell line was completely
hypomethylated. The band detected at
5 kb in uninfected cell lines
(lymphoid NK 3.3 and monocytoid
THP-1) digested with
BamHI
and
SnaBI is consistent with hypomethylation
of this site
(Fig.
4, groups 2 and 3). Interestingly,
in both
of the human retrovirally infected cell lines, the 5-kb band
was
greatly diminished or nonexistent and specific hybridization with
the full-length probe now occurred at 8.6 kb (Fig.
4, groups 1
and 4, respectively; Fig.
5B). These data
demonstrate that hypermethylation
of this methyl-sensitive site can
occur during HTLV-1 and HIV-1
infection in cell lines. Thus, infection
with both known pathogenic
human retroviruses correlates with altered
methylation patterns
of IFN-

in the infected cells.

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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.
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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.
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To analyze the methylation status of the IFN-

promoter in samples
where few cells were available (e.g., HIV-1-infected T
H1
clones and patient samples), we developed a PCR-based analysis
of the
methyl-sensitive
SnaBI site. The premise of this method
is
that DNA cut by
SnaBI will not yield a product with a PCR
primer
pair which spans the
SnaBI site (Fig.
5A). The
downstream primer
pair which does not contain a
SnaBI site
and should have a PCR
product was always included as a control for a
poor PCR. The method
was validated by the presence of a band in
T
H2 clones (Fig.
5C,
group 2), which do not express the
IFN-

gene and are methylated
at the
SnaBI site as shown
by Southern analysis (
84) and by
the lack of a PCR product
in T
H1 clones (Fig.
5C, group 1), which
express the IFN-

gene and are not methylated as determined by
Southern analysis. Acute
infection of susceptible lymphoid cell
lines with either HTLV-1 or
HIV-1 resulted in hypermethylation
of the
SnaBI site, as
demonstrated by detection of increased PCR
products (Fig.
5B; Fig.
5C,
groups 3 versus 4 and 5 versus 6).
We next examined whether increased DNA MTase expression correlated with
increased methylation of the IFN-

promoter and decreased
IFN-

mRNA expression and production following acute infection
by HTLV-1 and
HIV-1 in primary cells. Since acute infection of
primary
T
H1 clones by HIV-1 stimulated a marked increase in DNA
MTase expression (Fig.
1C), the methylation status of the IFN-
promoter in these HIV-1-infected T
H1 clones was analyzed by
PCR
(Fig.
6A) following acute infection.
A marked increase in the
methylation of the IFN-

promoter occurred
in HIV-1-infected T
H1
clones (Fig.
6A; Table
4). As shown by RT-PCR, IFN-

mRNA
expression
was not detectable at day 10 following HIV infection of the
T
H1
clones (Fig.
6B). In contrast, detectable levels of
IFN-

mRNA
were observed in the uninfected clones. Similarly, acute
HTLV-1
infection of NK 3.3, which produces IFN-

after stimulation
with
IL-2 (
80), resulted in increased DNA MTase expression
and concomitant
downregulation of IFN-

expression (data not shown).
In five individual
T
H1 clones, IFN-

expression and
production correlated with the
methylation status of the promoter
during HIV-1 infection of T
H1
clones in vitro (Table
4).

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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.
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To further strengthen the correlation between acute HIV infection,
increased MTase activity and downregulation of IFN-

, an
ELISPOT for
IFN-

was developed. This assay assesses production
of the cytokine
at the level of the individual cell and allows
determination of the
number of cells producing IFN-

in replicate
cultures following HIV
infection. Using three different T
H1 clones
(H1.12, H1.15,
and H1.18) whose IFN-

promoter is methylated during
HIV-1 infection
(Table
4), we found 67, 70, and 80% reductions,
respectively, in the
number of IFN-

-producing cells 2 days after
infection as shown by
ELISPOT. All five T
H1 clones showed greater
than a fivefold
increase in methylation of the IFN-

promoter.
Taken together, these
data indicate that the IFN-

gene is a target
for de novo methylation
as a result of the high levels of DNA
MTase activity following acute
HIV infection of primary lymphocytes.
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.

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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.
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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.
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Furthermore, when parental JMO cells were stably transfected with the
antisense MTase vector, the resulting cell line, JMO-TMH,
showed a
>90% reduction in DNA MTase expression (Fig.
8A) along
with
expression of the antisense mRNA (Fig.
8B) as determined
by RPA. When
IFN-

mRNA expression from these cell lines was measured
by RT-PCR,
we found that IFN-

mRNA was markedly higher in the
antisense-expressing line (Fig.
9A, lane
3) than in the control
cell lines stably transfected with neomycin
(JMO-neo) and sense
(JMO-HMT) constructs (Fig.
9A, lanes 1 and 2, respectively). The
reduction in the IFN-

mRNA in the sense
transfectants was threefold
when quantitated and normalized against the
glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) control. As previously
described (
75),
the overexpression of sense MTase was not
well tolerated by the
cells. These transfected cells either lost
overexpression of MTase
or died within 10 days and were not further
analyzed.

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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.
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Southern analysis showed partial methylation of the IFN-

promoter in
the control JMO-neo cell line (Fig.
9B, lanes 4 and
7), while JMO-TMH
was completely hypomethylated not only at the
SnaBI site
(Fig.
9B, lane 5) but also at the
HpaII site in the
first
intron, near the intronic enhancer elements (Fig.
5A; Fig.
9B, lane 8),
as evidenced by the generation of 4.9- and 3.8-kb
bands following
BamHI-
HpaII digestion. Expression of an antisense
MTase in the JMO cell line shows a direct effect of DNA MTase
activity
on IFN-

production. One hundred percent of cells carrying
the
antisense gene produced IFN-

, compared to 22% of parental
JMO as
measured by ELISPOT (Table
5). On a per-cell basis, the
antisense
containing cells produced 5- to 10-fold more IFN-

.
These
observations were similar for both the bulk culture and
single-cell
clones of JMO-TMH, indicating the results were not
due to cell-to-cell
variability (Table
5), even though there
is heterogeneity in basal
expression of MTase and IFN-

among
individual clones within the
population of parental JMO cells.
Thus, MTase expression and IFN-

gene expression are directly
linked in these cells. The ability to
modulate MTase and IFN-
by expression of antisense MTase allowed us
to directly test the
correlation between HIV-1 and MTase modulation of
IFN-

expression.
The D8 clone of JMO, expressing IFN-

in 36% of
the population
as determined by ELISPOT (Table
5), was infected for 7 days with
HIV-1. At this time, ELISPOT analysis detected only 20% of
the
cells expressing IFN-

. These cells were then transiently
transfected
with the antisense construct by using Superfect according
to the
manufacturer's protocol. At 48 h, the cells were harvested
and
ELISPOT was performed. The HIV-infected JMO population which had
20% expressing cells now had 36% of the cells expressing IFN-
compared to the same cells transfected with a control construct,
which
was unchanged at 20% expressing cells (Table
5). These
data indicate a
causal link between HIV-1 infection, MTase expression,
and the
downregulation of IFN-

expression.
 |
DISCUSSION |
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 TH1 and
TH2 helper T-cell subsets. TH1 cells, which
produce IFN-
, are hypomethylated at this site, while TH2
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
TH2 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
TH1 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 TH1
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 TH1
cells, downregulation of the production of TH2 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
TH2 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
TH1, methylation of the IFN-
promoter could lead to a low estimate of the number of TH1 cells in a population.
Second, decreased IFN-
expression could result in a decrease in
production of TH1 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
TH1-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
TH1 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.
 |
ACKNOWLEDGMENTS |
We thank Kathleen Wieman and Jason Troxell for excellent
technical assistance.
Portions of the work in the laboratory of S.B.B. were supported by NIH
grant CA43318 and in part with funds from the NCI, NIH, under contract
no. NOI-CO-56000.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: P.O. Box B,
Bldg. 567, Rm. 253, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201. Phone: (301) 846-5610. Fax: (301) 846-7034. E-mail:
Mikovits{at}fcrfv1.ncifcrf.gov.
 |
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Molecular and Cellular Biology, September 1998, p. 5166-5177, Vol. 18, No. 9
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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