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- But Not of IFN-
-Induced JAK/STAT Pathway in a Subset of U937 Clones Prevents the Antiretroviral Effect of IFN- Against HIV-11
*
AIDS Immunophatogenesis Unit, San Raffaele Scientific Institute, Milan, Italy; and
Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induces transcription of several IFN-stimulated genes
(ISGs). Recently, the IFN--dependent Janus kinase (JAK)/STAT pathway
has been shown to mediate the activation of some ISGs, by the
sequential phosphorylation of two JAK kinases (JAK1 and JAK2) and of
STAT1. Given that the JAK/STAT is the major, but not the only pathway
linked to the IFN-R, aim of our work was to investigate the
signal-transduction pathway(s) by which IFN- exerts its effects on
acute replication of HIV in monocytic cells. To this end, we utilized
clones previously derived from the U937 promonocytic cell line,
differing for their efficient (plus clones) or inefficient (minus
clones) abilities of supporting HIV replication. Unlike IFN-
,
IFN- did not inhibit HIV replication in plus clones, whereas virus
production in minus cells was efficiently inhibited by both types of
IFN. Plus clones generated a JAK/STAT signal-transduction pathway in
response to IFN-, but not IFN-. In contrast, minus clones
responded to either cytokines. The functional defect of plus clones in
response to IFN- was correlated to a selective defect of IFN-R2,
but not IFN-R1, membrane expression. Surprisingly enough, IFN-
stimulation of plus clones induced IFN-stimulated gene factor 3
(ISGF3). These results strongly support the hypothesis that the
JAK/STAT pathway is responsible for the antiretroviral effect of
IFN-, and further provide evidence for a potential second pathway
triggered by IFN- in the absence of IFN-R2 chain cell surface
expression and involving ISGF3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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has been shown to exert dichotomous effects on HIV infection in
vitro in both CD4+ T lymphocytes and monocytes/macrophages,
the two main cell types productively or latently infected by HIV
(reviewed in Refs. 2 and 3). Enhancement or suppression of viral
replication can be attributed either to the different time of
stimulation by the cytokine versus the time of infection, or to the
different experimental conditions and systems employed, such as
unfractionated PBMC, cell lines, or purified primary cells (2). In
primary monocyte-derived macrophages, HIV expression was enhanced or
repressed as a function of whether IFN- was added to the cultures
before or after infection, respectively (4), or whether serum from
HIV-positive individuals was present (5). The described effect of
IFN- on HIV life cycle in T cells ranges from enhancement (6) to
inhibition (7) or no effect (8). Recently, it has been shown that
endogenous IFN- enhanced HIV replication in CD4+ T
lymphocytes immortalized with herpesvirus saimiri (9) in agreement with
previous findings in IL-2-stimulated PBMC of most donors (10). In
contrast, exogenous stimulation by IFN- of T cell lines and primary
T lymphocytes has been shown to inhibit HIV production (7). Protection
against HIV-1 infection has been demonstrated in primary
monocyte-derived macrophages in endotoxin-free culture conditions (11, 12) and in acutely infected THP-1 and U937 monocytic cell lines (13, 14). However, IFN- stimulation of the chronically HIV-infected
U937-derived U1 cells promoted a clear-cut up-regulation of virus
production. This effect was masked by the redirection of the
predominant site of virion production from the cell surface to the
intracytoplasmic vacuoles under phorbol ester-stimulated conditions
(15).
In the face of this large body of literature, the molecular mechanisms
underlying the effect of IFN- on HIV-infected cells have not been
investigated to date. In this regard, IFN- exerts its actions by
activating a number of genes containing a specific
cis-acting element called IFN--activated sequence in
their promoter region that is necessary and sufficient to confer
IFN- responsiveness (16). The chain of events by which the signal
triggered by IFN- at the level of the plasma membrane can lead to
gene transcription has been studied extensively in the recent years
(reviewed in Refs. 17 and 18). This pathway, named
JAK/STAT,3 consists in a
cascade of sequential tyrosine phosphorylations that activate
cytoplasmic intermediates. Engagement of IFN-R by the ligand results
in the activation of the two receptor-associated JAK1 and JAK2 kinases
that in turn phosphorylate Tyr440 of the cytoplasmic domain
of the IFN-R1 chain that functions as docking site for the SH2
domain of STAT1. Receptor-bound STAT1s then become substrates of the
JAK kinases. Activated STAT1s dimerize, translocate to the nucleus, and
stimulate the transcription of ISGs, through the binding to
IFN--activated sequence. This pathway was first characterized for
the IFN-R (19), but since then additional members of the JAK and
STAT families have been identified as components of the
signal-transduction pathways of a variety of other cytokine receptors
(17, 18). In early studies, in addition to JAK/STAT pathway, protein
kinase C or ion fluxes have been linked to cell activation upon IFN-
stimulation (20).
In the present study, we have investigated the molecular mechanisms
involved in IFN- antiviral effects on U937 cells acutely infected
with HIV. In particular, we investigated the ability of IFN- and
IFN- to exert an antiviral effect in a group of U937-derived clones,
previously defined for their ability (plus clones) or inefficiency
(minus clones) in supporting to HIV replication (21, 22). We observed
that plus clones were resistant to the antiviral effect of IFN-, but
not to that of IFN-, and did not generate an IFN--induced
JAK/STAT signal-transduction pathway as a consequence of lack of
expression of IFN-R2. Furthermore, in this study we show evidence
suggesting the existence of a potential second pathway triggered by the
IFN-/IFN-R interaction that can signal in the absence of the
IFN-R2 chain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U937 SB-derived promonocytic plus (clone 10) and minus (clone
34) clones and their derived subclones were grown in RPMI 1640
supplemented with 2 mM glutamine, penicillin (100 U/ml), streptomycin
(100 µg/ml) (BioWhittaker, Verviers, Belgium), and 10% FCS (HyClone
Europe, Cramlington, U.K.). Clone 10- and clone 34-derived subclones
were obtained by limiting end-pointing dilution, as previously
described (22). Human rIFN- was purchased from R&D Systems
(Minneapolis, MN). rIFN-2 was purchased from Schering-Plough
(Kenilworth, NJ).
HIV infection
Plus and minus clones were acutely infected with
HIV-1IIIB/LAI strain (ABI, Advanced Biotechnologies,
Columbia, MD) at a multiplicity of infection (MOI) of approximately
0.1, resuspended in complete medium in the presence or absence of 1000
or 5000 U/ml of rIFN- or 100 or 500 U/ml of IFN-2, and then
seeded (1–2.5 x 105/well) in duplicate wells in
48-well polystyrene culture plates (Falcon; Becton Dickinson
Laboratories, Lincoln Park, NJ). Cells were counted every 4 days and
splitted weekly. Culture supernatants were harvested and stored at
-80°C until tested for a Mg2+-dependent reverse
transcriptase (RT) activity assay (21, 22).
Antibodies
Affinity-purified rabbit polyclonal Ab (sc-346) and mouse mAB
(sc-464) raised against STAT1/ß, affinity-purified rabbit
polyclonal anti-IRF1 Ab (sc-497), and anti-ISGF3 Ab (sc-496)
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA);
anti-STAT2 antiserum was a generous gift from X.-Y. Fu (Department
of Pathology, Yale University School of Medicine, New Haven, CT);
anti-JAK1 antiserum was a generous gift from A. Zimiecki
(Laboratory for Clinical and Experimental Cancer Research, University
of Berna, Switzerland); anti-Tyk2 antiserum (R5) and mAbs (T10-2)
were a generous gift from S. Pellegrini (Unit 138, Institut National de
la Santé et de la Recherche Médicale, 276, Institut
Pasteur, Paris, France); mouse anti-phosphotyrosine mAb 4G10
(05-321) and whole rabbit anti-JAK2 antiserum (06-255) were
purchased from Upstate Biotechnology (Lake Placid, NY); and mouse
anti-human IFN-R1 chain mAb (1223-01) was purchased from Genzyme
(Cambridge, MA), whereas mouse mAb C.11, an IgG2a that specifically
interacts with the extracellular domain of human IFN-R2 chain (23)
was kindly provided by S. Pestka (UMDNJ-Robert Wood Johnson Medical
School, Pitscaway, NJ) and G. Garotta (Human Genome Sciences,
Rockville, MD). Anti-MHC class I mAb B9-12 was kindly provided by R.
Accolla (CBA, Genova, Italy).
Northern blot and RT-PCR analyses
Total cellular RNA was extracted by the guanidine isothiocyanate
method using RNAzol B (Biotecx Laboratories, Houston, Texas) according
to the manufacturer’s instructions. For Northern blot analysis, 10
µg of total RNA for minus cells and 15 µg for plus cells were
separated in 1% agarose formaldehyde gel electrophoresis and
transferred to nylon membranes Hybond-N (Amersham Life-Science, Little
Chalfont, U.K.). RNAs were UV cross-linked in UV Stratalinker 1800
(Stratagene, La Jolla, CA) to the membranes that were then hybridized
overnight at 42°C in 50% formamide containing 10% dextran sulfate
with -32P-labeled probe. The probe, a 2-kb fragment of
human IRF1 cDNA cloned in the HindIII-NotI site
of pcDNA3 plasmid (Invitrogen, San Diego, CA), was labeled by Megaprime
DNA labeling system and [-32P]dCTP (3000 Ci/mmol)
(Amersham). After extensive washing, the membranes were exposed to
Hyperfilm-MP (Amersham) film. To verify that equal amount of total RNA
was loaded into the gels, filters were stripped and rehybridized with
-32P-labeled 1-kb fragment of the human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Membranes were
scanned by a PhosphorImager (Molecular Dynamics, Menlo Park, CA) for
quantification analysis.
For RT-PCR, total RNA (1 µg), extracted as mentioned above, was first
incubated for 5 min at 68°C, followed by 5 min at 94°C, and then
reverse transcribed by incubation at 37°C for 60 min in a 50-µl
reaction mix containing 1x RT buffer (Life Technologies, Paisley,
U.K.), 800 µmol/L dNTP (Pharmacia, Piscataway, NJ), 16 U rRNasin
(Promega, Madison, WI), 1 µg of random examers pd(N)6
(Promega), and 50 U of murine Moloney leukemia virus RT (Life
Technologies). Twenty-five microliters of a 1/100 dilution of the cDNA
preparation, obtained from the RT reaction, were amplified in a 25-µl
reaction mix containing 1x PCR buffer (Perkin-Elmer, Norwalk, CT), 2.5
mM MgCl2, 200 µmol/L dNTP, and 1.25 U AmpliTaq Gold
(Perkin-Elmer) in a GeneAmp PCR system 9600 thermal cycler
(Perkin-Elmer) with 50 and 20 pmol/L of cold primers specific for
ISGF3 (forward, 5'-CTG TGC TCC AGG ACT CCC TC; reverse, 5'-GGA AGC
AGA AAC TCC AGG GAC) and GAPDH, used as internal control (forward,
5'-CCA TGG AGA AGG CTG GGG; reverse, 5'-CAA AGT TGT CAT GGA TGA CC),
respectively. The antisense primer was end labeled with
[-32P]ATP and T4 polynucleotide kinase (Promega), and
the equivalent of 100,000–300,000 cpm was added to the reaction mix.
The size of the PCR products are 169 bp for ISGF3 and 195 bp for
GAPDH. PCR conditions for both ISGF3 and GAPDH were: 94°C for 10
min for denaturation, then 94°C for 30 s, 59°C for 45 s,
72°C for 1 min for 26 cycles, followed by 72°C for 10 min. PCR
amplification products were resolved on a 5% polyacrylamide gel
electrophoresis using 0.5x Tris-borate-EDTA as running buffer. The
gels were dried, exposed to x-ray films, and then scanned by
PhosphorImager for quantification analysis.
Cell extracts and electrophoretic mobility shift assay (EMSA)
Nuclear and cytoplasmic extracts were prepared according to a published procedure (24), with minor modifications. Briefly, both buffers A and C contained a mixture of protease inhibitors that included leupeptin (10 µg/ml), pepstatin A (10 µg/ml), aprotinin (33 µg/ml), E-64 (10 µg/ml), Pefabloc SC AEBSF (1 mM), and diisopropyl fluorophosphate (3 mM); and the phosphatase inhibitors sodium vanadate (Na3V04) (1 mM) and sodium fluoride (NaF) (50 mM). Following the lysis of the plasma membranes by vortexing the cells resuspended in buffer A containing 0.1% Nonidet P-40 for 10 s, nuclei were pelleted and the supernatants (cytoplasmic extracts) were recentrifuged at 12,000 x g for 15 min at 4°C, aliquoted, and stored at -80°C. Pelleted nuclei were resuspended in buffer C, and after a 30-min incubation on ice with occasional vortexing, samples thus treated were spun at 12,000 x g for 15 min at 4°C. The resulting supernatants (nuclear extracts) were aliquoted and stored at -80°C. Protein concentration determination of the nuclear and cytoplasmic extracts was evaluated by a protein assay kit based on the Bradford method (Bio-Rad, Hercules, CA).
For EMSA, nuclear extracts were incubated with a
[-32P]ATP end-labeled double-stranded oligonucleotide
corresponding to the IFN--responsive region (GRR) element located
within the promoter of the FcRI gene (5'-CTT TTC TGG GAA ATA CAT CTC
AAA TCC TTG AAA CAT GCT-3') (25), or with a double-stranded
oligonucleotide corresponding to the IFN-stimulated response element
(ISRE) located within the promoter of the ISG15 gene (5'-GAT CCT CGG
GAA AGG GAA ACC GAA ACT GAA GCC-3') (26), and the DNA-protein complexes
were resolved as previously described (27).
Immunoprecipitation and Western blot analyses
Cytoplasmic or nuclear proteins were incubated in 1 ml buffer
(40 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM
Na3VO4, 50 mM NaF) with 20 µl of a 50%
slurry of protein A agarose (Boehringer Mannheim, Mannheim, Germany) in
the presence of a 1/500 dilution of specific Abs: anti-STAT1
(sc-346), anti-JAK1, anti-JAK2, and anti-Tyk2 (R5) Abs.
Suspensions were incubated overnight at 4°C in a rotating wheel.
Immunoprecipitates were washed several times with 1 ml of washing
buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 1% Triton X-100, 0.1% SDS, 1
mM Na3V04, 50 mM NaF) before electrophoretic
separation on 7.5% SDS-PAGE and subsequent transfer to nitrocellulose
membrane Hybond ECL (Amersham) by electroblotting. For IRF1 and
ISGF3 immunoblotting, nuclear proteins were separated on 10%
SDS-PAGE. Membranes were blocked in 7.5% BSA, 20 mM Tris, pH 7.6, 137
mM NaCl, and 0.2% Tween-20 for 1 h at room temperature and
further incubated (overnight at 4°C) with the desired primary Ab.
Anti-STAT1 Ab (sc-464) was diluted 1/500, whereas anti-JAK1,
anti-JAK2, anti-Tyk2 (T10-2), anti-ISGF3, anti-IRF1,
and anti-phosphotyrosine 4G10 Abs were diluted 1/2,000. Ab binding
was visualized by using the appropriate horseradish
peroxidase-conjugated secondary Ab (anti-mouse or anti-rabbit
Abs, diluted 1/5,000 or 1/15,000, respectively). The signal was
revealed by the enhanced chemoluminescence system (ECL; Amersham,
Little Chalfont, U.K.) according to the manufacturer’s instructions.
Flow-cytofluorometric analysis (FACS)
The presence of cell surface IFN-R1 and IFN-R2 chains and
MHC class I was evaluated on plus and minus clones by cytofluorometric
analysis on a FACScan (Becton Dickinson, San Jose, CA) and analyzed by
a CellQuest software (Becton Dickinson). Plus and minus cells were
cultured at 2 x 105/ml in 24-well culture plates in
the presence or absence of IFN- (1000 U/ml). After 3 and 4 days of
culture, cells were harvested, counted, washed, and resuspended at
2 x 105 cells/tube on ice for phenotypic staining.
Common indirect immunofluorescence procedures were followed. Cells were
incubated with primary anti-MHC class I mAb B9-12 (1/200 final
dilution) or control mouse IgG1 Abs at 1 mg/tube (Jackson
ImmunoResearch, West Grove, PA) for 30 min on ice with the secondary
Ab, FITC-conjugated F(ab')2 fragment goat anti-mouse
IgG (1:100) (Jackson ImmunoResearch), washed twice with cold PBS, and
fixed with 1% formaldehyde before acquisition and analysis. The same
procedure was followed to stain untreated cells to monitor expression
of the IFN-R1 chain with a commercially available mAb (Genzyme). To
detect the surface expression of the IFN-R2, the primary mAb C11 was
followed by biotinylated rabbit anti-mouse IgG (1:100) (Dako,
Glostrup, Denmark) and then by phycoerythrin-conjugated streptavidin
(Dako).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on HIV-1 replication in plus and
minus cells
We have reported previously (21) that minus clones exhibit a
markedly delayed kinetics of acute HIV infection compared with the plus
clones. To investigate the molecular mechanisms of the antiviral effect
of IFN-, we used the two distinct groups of U937-derived clones as
experimental model. Cells were infected with HIV-1IIIB/LAI
strain at a MOI of 0.1 and either left untreated or treated with two
different concentrations (1000 or 5000 U/ml) of IFN-. Kinetics of
infection were followed for 
35 days. In agreement with previous
results (21), plus cells showed a peak of RT activity values between
day 10 and 15 postinfection, whereas the peak was delayed about 2 wk in
minus cells (Fig. 1
A).
Strikingly, we found that HIV replication was not inhibited in plus
cells, even at the highest concentration, by IFN-. In contrast, HIV
replication was inhibited in minus cells in a concentration-dependent
manner (Fig. 1A). Unlike minus cells, plus cells were also
insensitive to the antiproliferative effect of IFN-.
Concentration dependence was not observed in the
antiproliferative action of IFN- on cell growth of minus cells (Fig. 1B). Thus, plus cells appeared somehow incapable of
responding to IFN-.
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-dependent MHC class I expression in plus and minus cells
To examine whether plus cells were broadly unresponsive to IFN-
actions, we tested the expression of MHC class I surface Ag by
cytofluorometric analysis. In this regard, it is known that increased
expression of these glycoproteins at the plasma membrane level is
usually observed after a prolonged stimulation (48–72 h) with IFN-
(28). As shown in Fig. 2, both plus and
minus cells expressed constitutive levels of MHC class I Ags. However,
class I Ag expression was up-regulated after IFN- treatment
exclusively in minus cells (Fig. 2).
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-dependent IRF1 and ISGF3 protein expression in plus and
minus cells
We then analyzed the induction of IRF1 and ISGF3, two
transcription factors induced by IFN- stimulation earlier than class
I Ag expression. Western blot was performed with nuclear extracts from
plus and minus cells either untreated or treated for 17 h with
1000 U/ml of IFN-. Membranes were sequentially incubated with
polyclonal Abs raised against IRF1 and ISGF3 proteins (Fig. 3). IRF1 was induced over a very low
basal level (depending on clone variability) in minus cells, whereas
plus cells were unresponsive in terms of IRF1 induction (Fig. 3). In
contrast, when the same filter was stripped and reprobed with
anti-ISGF3 polyclonal Ab, both clones showed an equal
IFN--dependent induction over the low constitutive levels of
ISGF3 protein (Fig. 3). Induction of IRF1 in minus cells and of
ISGF3 in both types of clones was detectable as early as after
6 h of IFN- treatment, as later discussed.
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-dependent IRF1 and ISGF3 mRNA induction in plus and
minus cells
To further demonstrate the differential inducibility of IRF1 and
ISGF3 in plus and minus cells, we investigated the ability of
IFN- to modulate IRF1 and ISGF3 steady state mRNA levels (Fig. 4). Total RNA was extracted from cells
that were either unstimulated or stimulated with IFN- (1000 U/ml)
for 1 and 6 h, and IRF1 mRNA levels were evaluated by Northern
blot analysis using, as a probe, a human IRF1 full-length cDNA.
Consistent with the immunoblotting data, IRF1 mRNA level was increased
by IFN- treatment exclusively in minus cells (Fig. 4A).
Using as internal standard a GAPDH probe, we estimated 7- and 10-fold
increased levels of IRF1 mRNA after 1 and 6 h of IFN-
stimulation, respectively (not shown). ISGF3 mRNA levels were
evaluated by RT-PCR analysis because of its low abundance (Fig. 4B). As expected, ISGF3 mRNA was induced over its
constitutive level by IFN- in both types of clones. Taken together,
these observations strongly support the conclusion that plus cells are
not generally unresponsive to IFN-, although they are specifically
defective in mounting an antiretroviral response after stimulation by
this cytokine.
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-dependent STAT1 activation in plus and minus cells
To gain further insights into the differential ability of plus and
minus cells to respond to IFN-, we analyzed the IFN-R/JAK/STAT
pathway. Immunoprecipitation of cytoplasmic and nuclear proteins,
obtained from cells either unstimulated or stimulated with IFN- for
30 min, was performed using a rabbit polyclonal Ab raised against
STAT1. The immunoprecipitated STAT1s were probed with the
anti-phosphotyrosine mAb 4G10 by immunoblotting. As shown in Fig. 5A (upper panel),
STAT1 was activated in minus, but not in plus cells. The percentage of
activated STAT1 was higher in the nuclear compared with the cytosolic
fraction, indicating that nuclear translocation indeed occurred. In
contrast, STAT1 was present only in the nonphosphorylated form in the
cytosol, but not in the nucleus in plus cells (Fig. 5A).
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(1000 U/ml). STAT1 dimers and tetramers, whose specificity was
determined by Ab supershifting, bound to GRR element in minus cells
even after 6 h of stimulation, whereas only very low levels of
DNA-binding activity, barely detectable after 6 h, were seen after
30 min of stimulation in plus cells (Fig. 5
B).
IFN--induced JAK activation in plus and minus cells
We next determined the activation of JAK by immunoprecipitations,
followed by Western blotting using the 4G10 mAb (Fig. 6). Despite the fact that JAK1 protein
was present in both types of clones at comparable amounts, its
activated form was detected only in the minus cells (Fig. 6). Similar
experiments performed with an anti-JAK2-specific polyclonal Ab
produced similar results (Fig. 6) in that JAK2 was activated only in
minus cells. These data demonstrate that the impaired response to
IFN- signaling in plus cells is not accounted for the lack of STAT1
or JAK1/2 protein expression.
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R1 and IFN-R2 chain expression in plus and minus cells
We finally studied the membrane expression of the IFN-R1 and
IFN-R2 chains by FACS analysis using two different Abs that
independently recognize the two receptor chains (Fig. 7). Both minus and plus cells expressed
high levels of the IFN-R1 chain on their cell surface. In contrast,
expression of the IFN-R2 chain was found exclusively on the membrane
of minus cells (Fig. 7). Of interest, the lack of IFN-R2 chain
expression on plus cells was not due to a defect at the
transcriptional/translational level since staining of permeabilized
cells demonstrated high levels of IFN-R2 chain in the cytosol of
both minus and plus cells (data not shown). Thus, the deficient
JAK/STAT signal-transduction pathway in plus cells, responsible for the
anti-HIV effect of IFN-, is most likely explained by the lack of
expression of the IFN-R2 chain on the plasma membrane.
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on HIV-1 replication in plus and minus cells
To investigate whether the two distinct types of clones responded
differentially to the antiviral effect of type I IFN, cells were
infected with HIV-1IIIB/LAI strain at a MOI of 0.1 and
either left unstimulated or stimulated with two different
concentrations (100 or 500 U/ml) of IFN-. Unlike IFN-, HIV
replication was inhibited in both clones in a concentration-dependent
manner (Fig. 8A). The
antiretroviral effect of IFN- was maximal in minus cells at both
concentrations, whereas 30–50% inhibition of the peak of RT activity
was observed in plus cells at 100 U/ml and 500 U/ml of IFN-,
respectively.
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-dependent ISGF3/ISGF3 activation in plus and minus cells
To investigate the ability of plus and minus cells to respond to
IFN- in regard to the activation of the IFN--induced JAK/STAT
pathway, we analyzed the activation of ISGF3 by EMSA. ISGF3 is a
multiprotein complex constituted by IFN--tyrosine-phosphorylated
STAT1, STAT2, and by the DNA-binding subunit ISGF3 (16). ISGF3
promotes gene transcription by binding to the ISRE present in the
promoter of the IFN-ISGs (16). Nuclear extracts from cells
unstimulated or stimulated for 30 min with IFN- (500 U/ml) were
incubated with a radiolabeled ISRE probe. ISGF3 was induced in both
types of clones after 30 min of IFN- stimulation (Fig. 8B). The specificity of the binding was determined by
supershifting the complex with anti-STAT2 (Fig. 8B) and
anti-ISGF3 (not shown) Ab. To further elucidate the ability of
IFN- to induce not only the activation of ISGF3, but also the
synthesis of ISGF3, we performed Western blotting assay using
nuclear extracts from plus and minus cells unstimulated or stimulated
for 6 h with IFN- (500 U/ml). ISGF3 synthesis was induced in
both types of clones, although it was stronger in minus than in plus
cells over a low constitutive extent in both clones, even though to a
lower level compared with IFN- stimulation (Fig. 8C).
IFN--induced JAK activation in plus and minus cells
We finally analyzed the activation of JAK1 and Tyk2 kinases by
IFN-. JAK1 and Tyk2 are the two tyrosine kinases associated with the
intracellular region of the IFN-R (17). JAK1- and Tyk2-activated
proteins were immunoprecipitated by specific Ab and then detected by
Western blotting using the 4G10 mAb. JAK1 was highly activated in minus
cells; it was also activated by IFN- in plus cells, although weakly,
but clearly detectable over unstimulated cells. Similar experiments
performed with an anti-Tyk2-specific polyclonal Ab demonstrated
that Tyk2 was strongly activated in both plus and minus cells (Fig. 9). Surprisingly enough, Tyk2 was found
activated in minus cells even in unstimulated conditions. These results
clearly demonstrate that IFN- is able to inhibit HIV replication in
both types of U937 clones, although with a higher efficiency in minus
than in plus cells. Unlike what was observed with IFN-, the
IFN--dependent JAK/STAT pathway was not impaired in plus cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on acute HIV infection of human
monocytic cells requires the activation of the JAK/STAT pathway. In
addition, in this study we provide evidence that IFN-R can signal
even in the absence of IFN-R2 cell surface expression. By using U937
clones that efficiently (plus) and poorly (minus) support HIV
replication, we demonstrated that in plus, but not in minus cells,
IFN- was totally ineffective in inhibiting viral production. The
nature of the defect was the lack of activation of the
IFN--dependent JAK/STAT pathway in plus cells, most likely as a
consequence of the absence of IFN-R2 chain expression on the cell
surface. In contrast, U937 plus cells were responsive to IFN- both
in terms of antiviral effect and activation of the JAK/STAT pathway.
Thus, plus cells are specifically defective for the induction of the
IFN--dependent, and not of the IFN--dependent JAK/STAT pathway.
The existence of variability among U937 clones in terms of
susceptibility to HIV-1 infection and replication has been reported by
other investigators (29, 30). We have originally characterized plus and
minus U937 clones in respect to HIV-1 replication and linked their
patterns to the absence or presence, respectively, of a
cathepsin G-like protease cleaving the p65 subunit of the
nuclear factor-
B transcription factor complex (21). This enzymatic
activity, however, is generated during in vitro nuclear extraction (21)
and does not account for the inefficient replication of HIV-1 in minus
clones (31). In addition, we have observed that plus, but not minus,
U937 clones constitutively secrete TNF-, a well-described inducer of
nuclear factor-B-dependent HIV transcription (21), and that
neutralization of endogenous TNF- diminishes the kinetics of viral
replication in plus clones (P. Biswas, in preparation). Thus, multiple
and independent features are selectively associated with plus or minus
clones, most likely as a reflection of their different stage of
differentiation along the monocytic lineage.
In the present study, a selective defect in IFN- responsiveness was
correlated to the lack of cell surface expression of the IFN-R2
chain in plus cells. In this regard, the two chains of the IFN-R
seem to carry on different tasks in that the IFN-R1 chain is
required for the high affinity ligand binding (32), whereas the
IFN-R2 subunit is deputed to signal transduction (33, and reviewed
in Ref. 34). Our findings are in partial disagreement with this model
in that plus cells could signal, in a JAK/STAT-independent manner, in
the absence of cells surface expression of the IFN-R2 chain. The
result of this putative alternative signal-transduction pathway
activation was the induction of the transcription factor ISGF3 both
at the mRNA and at the protein level. In this regard, it has been shown
recently in the murine system that the expression of ISGF3 gene by
IFN- is dependent on a novel IFN--activated response element,
named -activated transcriptional element, which binds two novel
transcriptional activators named IFN--inducible factors (35). With
the caveat of comparing two different species systems, these findings
support our results showing that STAT1 activation is not necessary for
ISGF3 induction in U937 plus cells. ISGF3 and IRF1 can bind to the
same responsive element ISRE in the promoter region of the genes
induced by type I IFN (IFN-/ß) (16). However, a potential role for
ISGF3, the DNA-binding subunit of ISGF3, also in the IFN-
response, has been suggested previously (36, 37). The two transcription
factors, IRF1 and ISGF3, do not appear to play redundant roles in
the responses triggered by either class I or class II IFNs, as
demonstrated by the fact that some ISGs are uniquely regulated by
either ISGF3 (ISGF3), IRF1, or both in double knockout (KO) mice
(IRF1-/- and ISGF3-/-) (38).
IFN--mediated induction of 2'-5' oligoadenylate synthetase and
double-stranded RNA-dependent protein kinase genes are ISGF3
dependent, whereas IFN--mediated induction of the guanylate-binding
protein gene is IRF1 dependent (38). In this study, we show that
ISGF3 is equally expressed on both types of clones stimulated by
IFN- and IFN-, whereas IRF1 is weakly expressed by IFN- in
plus and minus cells and selectively expressed after IFN-
stimulation only in minus, but not in plus cells. This finding suggests
that ISGF3 may not be involved in mediating the antiretroviral
action of IFN- on HIV replication. Given that it has been postulated
that the 2'-5' oligoadenylate synthetase activity might be responsive
for the inhibition of HIV replication (3), we are currently
investigating whether this gene is induced in plus cells by
IFN-.
The possibility that a third putative chain of the IFN-R might
contribute to the formation of an active IFN-R complex in plus cells
is consistent with our results. The existence of an additional
accessory factor has been postulated previously based on the
demonstrations that transfection of hamster cells expressing the
human IFN-R2 with a vector carrying the human
IFN-R1 chain did not confer full protection against vesicular
stomatitis virus (VSV) after IFN- treatment (39), suggesting that
the IFN-R1 and IFN-R2 chains are not sufficient to account for
all of the biologic responses induced by IFN-. Very recently,
Petricoin et al. (40) have shown that activation of the tyrosine
phosphatase CD45 and of Lck and ZAP-70 tyrosine kinases, which are
components of the TCR signaling pathway, is required for the
antiproliferative effect, but neither for the activation of the
JAK/STAT pathway nor for the protection from measles virus infection
induced by IFN- in Jurkat cells. These findings corroborate the
hypothesis that also in the case of class II IFN, additional
intermediate components besides the JAK and STAT family members might
be involved in the signal-transduction pathway.
Lack of response to IFN-, due to the inability to express membrane
IFN-R2 chain, occurs, to our knowledge, only in human (22) and mouse
(41) T cells. In the mouse system, IFN- down-regulates the
expression of the IFN-R2 chain, producing a desensitization effect
in Th1 CD4+ T lymphocytes. In the human system, the absence
of IFN-R2 on the membrane of human Th1 lymphocytes is due to its
preferential intracellular expression rather than from its
IFN--induced down-modulation (42). Furthermore, human T cells
stimulated through the TCR and expanded in IL-2 are unresponsive to
IFN- because of the lack of IFN-R2 chain expression (43). Our
results suggest that a similar phenomenon may occur also in cells of
monocytic origin. However, since plus, as well as minus cells, do not
express (as assessed by RT-PCR) or secrete constitutively IFN- (data
not shown), and since high levels of IFN-R2 are present in the
cytosol of plus cells, it is likely that the absence of the IFN-R2
on the plasma membrane results from an alteration of its intracellular
trafficking (37).
Previous studies reported that activation of the JAK/STAT
signal-transduction pathway is required for the antiviral effect of
IFN- against several viruses. The involvement of the transcription
factor IRF1, whose induction by IFN- is STAT1 dependent, is critical
for the inhibition of the encephalomyocarditis virus (EMCV), and
Newcastle disease virus infection. Furthermore, cells overexpressing
IRF1 are resistant to EMCV, and Newcastle disease virus and also VSV
(44). In addition, IRF1 KO mice are more sensitive than wild type to
infection by EMCV, but not by herpes simplex virus and, in contrast to
the results obtained with the IRF1-transgenic cells, by VSV (45).
Similarly, STAT1 KO mice are more susceptible to VSV and mouse
hepatitis virus infection (46, 47). Thus, both STAT1 and IRF1 seem to
play a crucial role in the host defense against several viruses. It is
noteworthy that none of the viruses studied until now, whose
replication is inhibited by IFN- through activation of the JAK/STAT
pathway, belong to the retrovirus family. Thus, our observations
represent the first demonstration of a direct involvement of the
JAK/STAT activation pathway in the suppressive effect of IFN- on HIV
replication.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chiara Bovolenta, P2/P3 Laboratories, San Raffaele Scientific Institute, Via Olgettina n. 58, 20132 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: JAK, Janus kinase; EMCV, encephalomyocarditis virus; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRR, IFN--responsive region; IRF1, IFN-regulatory factor 1; ISG, IFN-stimulated gene; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated response element; KO, knockout; MOI, multiplicity of infection; RT, reverse transcriptase; VSV, vesicular stomatitis virus.
Received for publication May 26, 1998. Accepted for publication September 15, 1998.
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