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IMMUNOBIOLOGY
From the Laboratory of Virology, Istituto Superiore di
Sanità, Rome, Italy; Department of Biology, University of Roma
Tre, Rome, Italy; and Institute of Biomedical Technology, CNR, Rome,
Italy.
Monocytes/macrophages play a predominant role in the immunologic
network by secreting and reacting to a wide range of soluble factors.
Human immunodeficiency virus (HIV) infection leads to deep immunologic
dysfunctions, also as a consequence of alterations in the pattern of
cytokine release. Recent studies on in vivo models demonstrated that
the expression of HIV Nef alone mimics many pathogenetic effects of HIV
infection. In particular, Nef expression in monocytes/macrophages has
been correlated with remarkable modifications in the pattern of
secreted soluble factors, suggesting that the interaction of Nef with
monocytes/macrophages plays a role in the pathogenesis of acquired
immunodeficiency syndrome (AIDS). This study sought to define possible
alterations in intracellular signaling induced by Nef in
monocytes/macrophages. Results demonstrate that HIV-1 Nef specifically
activates both The pathology of acquired immunodeficiency syndrome
(AIDS) should be considered as the sum of effects generated by human
immunodeficiency virus (HIV) replication, with direct T-cell
destruction, and by a deep alteration in the pattern of soluble
factors. The latter largely depends on the extent of viral replication,
because it is hindered by effective antiretroviral therapy. HIV/simian
immunodeficiency virus (SIV) Nef, a multifunctional 27- to 34-kd
protein expressed early in viral replication, was demonstrated to be a
strong candidate for many of the pathogenic effects of HIV/SIV. This
was first highlighted by the evidence that monkeys failed to develop
the disease on infection with nef-deleted SIV,1
and later confirmed by the observation that nef transgenic
mice developed a syndrome strictly related to AIDS.2
Furthermore, Nef perturbs the pattern of secreted factors in different
cell types. As an example, the engagement of Nef with the We were interested in investigating whether the expression of Nef
influences the activation of signal tranducers and activators of
transcription (STAT) molecules (reviewed in Schindler and
Darnell,10 Leaman et al,11
Darnell,12 Stark et al,13 and Bromberg and Darnell14). STAT-governed pathways were first described by
Darnell and colleagues by studying the interferon (IFN)-induced
intracellular signal transduction.15 Seven different STATs
have been characterized so far. Activation of STATs is involved in the
response of a wide number of cytokines, growth factors, and hormones.
Typically, binding of cytokines with specific receptors lacking
intrinsic kinase activity in its cytoplasmic tail induces receptor
aggregation and recruitment of members of Janus kinases. These become
activated by phosphorylating themselves and tyrosine residues of the
receptor cytoplasmic tails. The receptor phosphotyrosines serve as
docking sites for the binding of inactive STAT through the Src-homology 2 (SH2) domains. STAT monomers become phosphorylated at a constant tyrosine residue and dimerize. The activated dimers translocate to the
nucleus thereby binding to specific DNA response elements, ultimately
influencing gene expression programs. Hence, alterations in the STAT
pathways could substantially influence cellular homeostasis. Few
reports regarding the effects of HIV-1 infection on STAT
activation/induction have been published. Pericle and coworkers
reported diminished levels of STAT-1 We were interested in establishing the contribution of
monocytes/macrophages in STAT activation induced by HIV-1.
Monocytes/macrophages play a crucial role in the defense against
microbial agents, and their altered behavior is considered a hallmark
of AIDS.18,19 Monocytes/macrophages replicate
preferentially HIV strains (defined as M-tropic) more represented in
the early phases of disease20,21 but are resistant to the
cytocidal effect of HIV, thus representing a reservoir for HIV
infection.22 Long-lasting detrimental effects induced by
released HIV and by an altered pattern of secreted soluble factors
assign to infected monocytes/macrophages a central role in the
development of AIDS pathogenesis.
Here, we demonstrate that the expression of either the whole viral
genome or the Nef protein alone is able to activate both STAT1- Cell cultures
Plasmids, virus preparations, infections, and detection
Pseudotyped HIV-1 virus preparations were obtained from supernatants of 293 cells 48 hours after cotransfection with different NL4-3 derivative and VSV-G-expressing plasmids (molar ratio 5:1) performed by the calcium phosphate method.27 Supernatants were clarified and concentrated by ultracentrifugation as described.28 Virus preparations were titrated by measuring HIV-1 p24 contents by quantitative enzyme-linked immunosorbent assay (ELISA; Abbott, Abbott Park, IL). Ten nanograms per 106 cells of pseudotyped HIV-1 were used to infect 7-day-old MDMs. Percentages of cells expressing intracytoplasmic HIV-1 Gag-related products were evaluated by FACS analyses after treatment with Permeafix (Ortho Diagnostic, Raritan, NJ) for 30 minutes at room temperature and labeling for 1 hour at room temperature with a l:50 dilution of KC57-RD1 PE-conjugated anti-HIV-1 Gag mAb (Coulter, Hialeah, FL). Reverse transcriptase assay was performed as described.29 Recombinant protein preparation and immunodepletions The Nef and Uvp-1 recombinant (r) proteins were obtained as 6 × His-tagged fusion proteins as previously described.23,30 As for rNef, the gene from the NL4-3 HIV-1 strain24 was amplified by polymerase chain reaction (PCR) and cloned in-frame with the 6 × His tag into the 5' BamHI/3' SalI sites of pQE 30 vector (Qiagen, Chatsworth, CA). rNef was purified by lysing bacteria in an 8-M urea buffer, and using Ni-NTA resin (Qiagen) following the manufacturer's recommendations. rNef was eluted stepwise with 100, 250, and 500 mM imidazole, and the fractions were collected and analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rNef-containing fractions were pooled and dialyzed stepwise against gradually decreasing concentrations of urea (ie, 3-fold dilutions, 24 hours' dialysis each) in 1 × phosphate-buffered saline (PBS) to both completely remove the denaturing agent and allow a correct refolding of the protein. Finally, 10-µg aliquots of rNef were analyzed by SDS-PAGE and found devoid of nonspecific bands through Coomassie brilliant blue staining. Recombinant protein preparations were scored as negative for the presence of bacterial endotoxins by using the Limulus amebocyte lysate assay (Biowhittaker, Walkersville, MD). To ensure a total and specific depletion of rNef, complete medium supplemented with 100 ng/mL rNef was incubated for 8 hours at 4°C with a 1:50 dilution of a cocktail containing 6 different monoclonal and polyclonal anti-Nef antibodies (all obtained from the National Institutes of Health AIDS Research and Reference Program). As a control, rNef-complemented medium was incubated with equal amounts of irrelevant isotype- and species-matched antibodies. Then, immunocomplexes were reacted with detergent-free protein A-G agarose beads (Pierce, Rockford, IL) overnight at 4°C. Afterward, immunocomplexes bound to protein A-G agarose were discarded through centrifugation; supernatants were filtered (0.22-µm pore diameter) and added to MDM cultures. MIP-1 and IL-10 amounts in supernatants
of rNef-treated MDMs were measured by ELISA (R & D Systems,
Minneapolis, MN). MIP-1 and IL-10 immunodepletions were performed as
for rNef by using specific neutralizing mAbs (R & D Systems). Checks
for the complete clearing of soluble factors were carried out by ELISA.
Western blot assay The MDMs were washed twice with PBS pH 7.4 and lysed in 20 mM HEPES pH 7.9, 50 mM NaCl, 10 mM EDTA, 2 mM EGTA, 0.5% nonionic detergent IGEPAL CA-630 (Sigma), 0.5 mM dithiothreitol (DTT), 20 mM sodium molibdate, 10 mM sodium orthovanadate, 100 mM sodium fluoride, 10 µg/mL leupeptin, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) for 20 minutes in ice. Whole-cell lysates were centrifuged at 6000g for 10 minutes at 4°C and the supernatants frozen at 80°C. The protein concentration of cell extracts was determined by
the Bio-Rad (Hercules, CA) protein assay. Aliquots of 30 µg cell
extracts were resolved on 7% to 10% SDS-PAGE and transferred by
electroblotting on polyvinylidene difluoride (PVDF; Immobilon-P, Millipore, Bedford, MA) membranes for 60 minutes at 100 V with a
Bio-Rad transblot. For the immunoassay, PVDF membranes were blocked in
3% bovine serum albumin (BSA) fraction V (Sigma) in TTBS/EDTA (10 mM
Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% Tween-20) for 30 minutes at
room temperature, then incubated for 1 hour at room temperature with
specific antibodies diluted in 1% BSA/TTBS-EDTA. Antibodies used in
the different immunoblotting were the following: polyclonal
anti-phosphotyrosine STAT1, polyclonal anti-human phosphoserine STAT1, and monoclonal anti-phosphotyrosine STAT5 A-B from UBI (Lake
Placid, NY); monoclonal anti-STAT1 and monoclonal anti-STAT5 from Transduction Laboratories (San Diego, CA); polyclonal
anti-interferon regulatory factor 1 (IRF-1) from Santa Cruz
Biotechnology (Santa Cruz, CA); and monoclonal anti- -tubulin from
ICN Biomedicals (Costa Mesa, CA). Immune complexes were detected with
horseradish peroxidase-conjugated goat anti-rabbit (Calbiochem,
Darmstadt, Germany) or goat anti-mouse (NEN, Boston, MA) antiserum
followed by enhanced chemoluminescence reaction (ECL; Amersham
Pharmacia Biotech, Milan, Italy). The efficiency of rNef
immunodepletions was checked on the protein A-G agarose fraction by an
anti-Nef quantitative Western blot analysis performed by running in
parallel decreasing amounts of rNef. This assay allowed us to detect
less than 100 pg recombinant protein (not shown).
DNA electrophoretic mobility shift assay Whole-cell extracts as prepared for Western blot assays were used. To measure the association between DNA binding proteins and different DNA sequences, the double-stranded oligonucleotides (12 pmol) described below were end-labeled with [32P]ATP (1.11 MBq, 222 TBq, NEN) by T4 polynucleotide kinase (Biolabs, Beverly, MA). The labeled oligonucleotide probes (74-130 mBq) were
incubated for 1 hour at 4°C and 20 minutes at room temperature in a
final volume of 20 µL containing 20 µg cell extract proteins prepared as above in a binding buffer containing 20 mM Tris (pH 7.5),
75 mM KCl, 1 mM DTT, 6 µg/mL BSA, 2 µg/mL poly(dI)-poly(dC) (Sigma), and 13% glycerol. To control the specificity of the
DNA-protein binding, cold competitors were added in 200-fold molar
excess of the radiolabeled probe. Antibody treatment for supershifts was performed by adding 1 µg specific antibodies to 20 µg cell extract proteins. In this regard, anti-STAT1 mAbs from Transduction Laboratories, and anti-STAT2, -STAT3, -IRF-1, and -p48 polyclonal antibodies from Santa Cruz Biotechnology were used. Cell extract proteins (10 µg) from HeLa cells treated with 100 IU/mL human rIFN- for 30 minutes were used as positive controls for the
detection of STAT1- and STAT3-containing complexes. Cell extract
proteins (20 µg) from HeLa cells treated with 500 IU/mL human
rIFN- for 2 hours were used as controls for detecting complexes
containing IFN--stimulated gene factor 3 (ISGF-3) and IRF-1. The
analysis of the DNA-protein complex was carried out on 5%
nondenaturating gel (acrylamide/bisacrylamide 29:1) in 1 × TBE buffer
pH 8.3 (100 mM Tris, 97 mM boric acid, 2.5 mM EDTA).
The following double-stranded oligonucleotides were used:
HIV-1 infection induces STAT1 activation in human MDMs: role of env and nef genes Activation of STATs in PBMCs infected either in vivo or in vitro by HIV-1 has been recently described.17 We attempted to define the contribution of monocytes/macrophages in such an activation as well as the HIV-1 proteins involved. Usually, specific STAT activation occurs in a very short time (ie, 15-30 minutes) after cell treatment with the stimulating factors. Afterward, overcoming of specific inhibitors gradually switches off STAT activation.34 Thus, to appropriately monitor and characterize possible effects on STAT activation of HIV-1 infection, a protocol of highly efficient, single-cycle HIV infection was required. Because in vitro infection of MDMs with even high multiplicity of infection (MOI) of wild-type HIV-1 leads to the virus expression in a minority of challenged cells,35,36 we decided to infect MDMs with HIV-1 preparations pseudotyped with the membrane glycoprotein from VSV (VSV-G). In our hands, a viral inoculum containing 10 ng HIV-1 p24 was sufficient to infect 106 cells with an efficiency of more than 95%. This was demonstrated through the intracellular FACS analysis for the expression of HIV-1 Gag-related products performed 24 hours after the infection (Figure 1A).
In such experimental conditions, the infection of MDMs with full-length
HIV-1 induces activation of STAT1. STAT1 exists as 2 protein products:
the 91-kd ( We conclude that HIV-1 infection is indeed able to activate STAT1 in MDMs and that this activation correlates with the expression of nef and env genes. Soluble recombinant Nef mimics the effects of the viral-encoded protein The Nef-dependent STAT1 activation appeared new and of potential great interest, whereas STAT1 activation as a consequence of the action of HIV-1 Env gp120 has been described in cells strictly related to monocytes/macrophages.39 It is conceivable that an alteration of the STAT pathway could be part of the contribution of Nef in the development of AIDS pathogenesis.We were able to reproduce STAT1 activation by adding recombinant
soluble Nef (rNef) to MDM cultures. We recently demonstrated that rNef
is able to enter primary human MDMs, thereby inducing effects
superimposable to those described for ectopically expressed Nef.23 We treated 7-day MDMs from different healthy donors
with 100 ng/mL rNef and analyzed STAT1 activation through Western blot at different times (Figure 2A). We
observed STAT1-
The specificity of the rNef effect was first demonstrated by treating MDMs with the rNef-complemented medium after Nef immunodepletion (Figure 2B). The complete rNef removal was checked by Western blot (not shown). Also, we did not detect STAT1 activation in MDMs treated with 100 ng/mL recombinant Uvp-130 (Figure 2C), a plasmid DNA invertase-resolvase protein recovered through an experimental procedure identical to that used for rNef production. Hence, we could formally exclude that STAT1 activation was a consequence of the presence of even undetectable amounts of products contaminating rNef preparations. Extracellular Nef has been detected in the majority of AIDS patients' sera analyzed at a concentration of 5 to 10 ng/mL.40 To define whether rNef-induced STAT1 activation observed in MDMs could have an in vivo relevance, we analyzed STAT1 activation in MDMs treated with rNef doses from 0.1 to 100 ng/mL. As shown in Figure 2C, STAT1 was strongly activated starting at a concentration of 10 ng/mL, pointing out the possibility that our observations reflect phenomena actually occurring in vivo. It has been reported that HIV-1 infection of PBMCs leads to
activation of a truncated form of STAT5 (
Characterization of rNef-induced STAT1 activation in MDMs We characterized the rNef-induced STAT1 activation in terms of the ability to bind specific target sequences. In particular, we tested by electrophoretic mobility shift assay (EMSA) the binding with the GAS element from IRF-1 promoter and the SIE of c-fos promoter. It has been described that GAS element binds STAT1 homodimers,10-15 and that SIE, a GAS-like element,41 binds either STAT1-1 homodimers, STAT3 (ie, a member of the STAT family having target sequences similar to those recognized by STAT1) homodimers, and STAT1-3 heterodimers.42Figure 4 shows EMSA carried out on total
cell extracts of MDMs. We detected the formation of a single complex
with both sequences tested. Cells infected with
Besides the phosphorylation at the Tyr701 residue, phosphorylation at
the Ser727 of activated STAT1-
To assess whether the rNef-induced STAT1 activation leads effectively
to gene activation, we tested the expression of IRF-1 protein, a
transcription factor member of the IRF family of transcriptional regulators (reviewed by Mamane et al46) encoded by a gene
transcriptionally regulated by STAT1 activation.47 MDMs
were infected with
IRF-1 binds specific elements (ie, ISRE and positive regulatory domain
1) present in the promoter of, respectively, IFN-regulated genes and
IFN- These data, together with the evidence that increased amounts of STAT1 (whose gene seems to be induced by activated STAT147) have been observed on rNef treatment (Figure 2A), strongly support the idea that Nef-induced STAT1 activation results in transcriptional induction of STAT1 target genes. STAT1 is activated through the release of soluble factors STATs are typically activated as a consequence of the JAK phosphorylation that follows the engagement of cytokines or growth factors to the specific receptors. The evidence that a regulatory viral protein apparently not recognizing a secretory pathway is able to activate the STAT1 signaling appeared at first glance intriguing. In fact, we already reported that rNef apparently does not recognize specific receptors on the membrane of human monocytes/macrophages.23 To establish whether Nef acts through an intracellular pathway or, differently, the Nef-induced STAT1 activation depended on the release of soluble factors, supernatants from MDMs treated for 2 hours with rNef were added to fresh MDMs from the same donors and STAT1 activation assayed shortly thereafter (ie, 30 and 60 minutes). Importantly, supernatants were tested only after immunodepletions that efficiently cleared the rNef originally added. Figure 7A shows that MDMs treated with Nef-immunodepleted medium conditioned by rNef-treated MDMs ( rNef CM)
efficiently activated STAT1. This indicates that soluble factors
induced by Nef are responsible for the STAT1 activation. The
fact that the activation occurs more rapidly (ie, 30 minutes) than
following rNef treatment (2 hours) further excludes that it depended on the presence of residual rNef after immunodepletion. Of note, STAT1
activation was observed also by treating for 1 hour fresh MDMs with
supernatants collected 8 hours after MDM infections with wild-type,
nef-, or env-deleted pseudotyped HIV-1, but not with the env/nef double-defective HIV-1 (Figure 7B). The
STAT1 activation observed in MDMs treated with supernatants from
nef HIV-1-infected MDMs likely originated at least in
part from HIV-1 Env gp120 shed from infected cultures.39
In these experiments, virus particles residual from challenging
inoculum were cleared from supernatants of infected MDMs by
ultracentrifugation. The effective removal of virus particles was
checked by reverse transcriptase assay (not shown).
We also attempted to identify the Nef-induced soluble factor(s)
responsible for the STAT1 activation. It was reported that either the
ectopic expression of Nef or the rNef treatment of human MDMs induces
the release of cytokines49,50 and
chemokines.4,23 Among such soluble factors, those known to
activate STAT1 are MIP-1
Active protein synthesis is required for the rNef-induced STAT1 activation Here, we show that MDMs respond to the presence of Nef by releasing soluble factors activating STAT1. We investigated the mechanism through which Nef induces the release of the STAT1-activating factor. It is conceivable that Nef induces a proteolytic maturation of a presynthesized factor, as occurs in IL-1 secretion. Alternatively, Nef could induce a de novo synthesis of the STAT1-activating factor. In this respect, we analyzed the rNef-dependent STAT1 activation in the presence of an inhibitor of the active protein synthesis. We pretreated MDMs with 5 µg/mL cycloheximide, and, after 2 hours, rNef was added for 2 additional hours. As control, cells were also treated with 100 U/mL human IFN-, inducing a cycloheximide-independent STAT1
activation. We analyzed by Western blot the STAT1 Tyr701 phosphorylation, as well as the levels of STAT1, IRF-1, and -tubulin proteins. As shown in Figure 9,
cycloheximide treatment abolished the ability of rNef, but not of
IFN-, to activate STAT1. The total expression of STAT1 was slightly
affected by cycloheximide treatment and tubulin levels were not
influenced, whereas IRF-1 expression dropped dramatically, as expected
in view of its short half-life (about 30 minutes).54
This result demonstrates that active protein synthesis is required for the autocrine-regulated, rNef-dependent STAT1 activation.
The use of a highly efficient, single-cycle infection protocol allowed us to demonstrate that HIV-1 infection of MDMs leads to the activation of both STAT1 isoforms. By using HIV-1 deletion mutants, the independent involvement of both HIV-1 nef and env gene expression in STAT1 activation has been established. Essentially overlapping results were obtained either by infecting MDMs with Nef expressing HIV-1 or by exploiting the ability of monocytes/macrophages to internalize rNef.23 This latter represents a useful tool for a more detailed characterization of the Nef-dependent activation of STATs and of its mechanism. In addition, this model seems to be relevant in that we demonstrated STAT1 activation by treating MDMs with rNef concentrations close to those detected in the serum of the majority of patients with AIDS (ie, 5-10 ng/mL).40 We observed that the extent of STAT1 activation diminished over the time in both rNef-treated and HIV-1-infected cells; the intracellular Nef levels in the latter case remained unaltered at all times considered (not shown). This is likely the consequence of either the raising of STAT-specific inhibitors,14,34,37 or of a block in the induction of the still unidentified Nef-dependent STAT1-inducing soluble factor. Nef induced STAT1- Nef-dependent STAT1 activation has been observed by adding supernatants from rNef-treated MDMs to fresh MDMs and overlapping results were obtained by using supernatants from MDMs infected by Nef-expressing HIV-1 strains. These data are consistent with the hypothesis that STAT1 activation largely depends on the effects of soluble factors released on Nef stimulation. However, we cannot formally exclude that at least part of STAT1 activation could be mediated by an endogenous mechanism. The fact that Nef-dependent STAT1 activation appears to be a cycloheximide-sensitive event implies that the presence of Nef induces an ex novo synthesis of the factors directly or indirectly activating STAT1. We excluded that release of MIP-1 It is well established that Nef can interact through its SH3 homology domain with cellular Src kinases, such as Lck (in T cells)55 and Hck (in monocytes/macrophages).56,57 Also, the interaction of Nef with Vav, leading to the activation of JNK/SAPK pathway, has been recently described.58 It should be of interest to establish whether some of the already described interactions are involved in the reactions leading to the synthesis and release of STAT1-activating soluble factors. As for the significance of the Nef-induced STAT activation in terms of AIDS pathogenesis, it has been described that infection by different viruses or even the expression of single viral proteins could generate an altered pattern of STAT activation. In particular, it has been reported that Epstein-Barr virus,59 Friend spleen focus-forming virus,60 or human hepatitis B virus X protein61 activates cell signaling cascades involving JAK/STAT pathways. Also for HIV-1, previous reports demonstrated an altered pattern of STAT1 and STAT5 response as a consequence of the viral infection.16,17,39 Even if it is known that STAT1 could be activated by several cytokines, experiments with knock-out mice revealed that STAT1 activation seems mainly involved in the response to type I and II IFNs.62,63 Thus, alterations in STAT1 biochemical pathways induced by Nef in monocytes/macrophages may lead to both a hyporesponsiveness to a possible anti-HIV effect of type I IFN and a dysregulation in the response to type II IFN, with an overall decay in the immune reactivity toward the infection. Results reported here suggest that the effects of HIV-1 infection on STAT5 activation, formerly observed in PBMCs,17 seem to involve mainly lymphocytes or viral products other than Nef. As indicated by studies performed with constitutively activated mutants, STAT5 promotes cell cycle progression and survival64 and contributes to factor-independent cell growth.65 Clearly, inappropriately prolonged cell survival of HIV-infected lymphocytes could favor HIV spread and diffusion, thus strongly influencing the progression of AIDS pathogenesis. An unresolved debate has developed around the role that Nef plays in the development of AIDS in humans. In fact, although the use of both SIV-infected Macaca and transgenic mouse systems generated consistent conclusions on the critical role of Nef in AIDS pathogenesis,1,2 much more controversial data regarding the role that HIV-1 Nef expression plays in humans have been published.66-70 We have already described how the internalization of extracellular Nef in MDMs could induce a preferential replication of T tropic, which is the HIV quasi species more represented in the later stages of the disease,22 compared to M-tropic HIV variants.23 Data presented here describe an additional consequence of Nef expression, that is, the alteration of the pattern of soluble factors released by monocytes/macrophages, which, in turn, could lead to modifications in the cross-talk between monocytes/macrophages and cells of the immune system. Our results indicate that Nef induces similar effects in MDMs when expressed endogenously or on extracellular protein internalization. This supports the hypothesis that in vivo Nef acts similarly either in cells infected by Nef-expressing HIV strains, or in monocytes/macrophages picking up extracellular Nef possibly released by apoptotic, HIV-infected lymphocytes in those tissue districts (ie, lymph nodes) where high levels of HIV replication couple with tight interactions between monocytes/macrophages and lymphocytes. The effects of Nef expression deserve great interest considering that monocytes/macrophages resist the cytopathic effect of HIV infection and, hence, an altered soluble factor pattern may influence the immune system as long as infected monocytes/macrophages survive HIV infection. Taken together, our data support the concept that Nef expression might induce detrimental effects in both infected and uninfected, neighboring cells. In addition, our model seems useful for the individuation of soluble factors involved in AIDS pathogenesis, as well as for new intracellular partners or functions of Nef in monocytes/macrophages.
This paper is dedicated to the memory of the late Professor Giovanni Battista Rossi and of the late Professor Franco Tatò. Monoclonal and polyclonal anti-Nef were obtained from the AIDS Research and Reference Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. We thank Drs M. Gabbianelli and E. Pelosi, Istituto Superiore di Sanità, Rome, for kindly providing peripheral blood mononuclear cell preparations. We are indebted to A. Lippa and F. M. Regini for the excellent editorial assistance.
Submitted March 12, 2001; accepted June 28, 2001.
Supported by grants from the AIDS project of the Ministry of Health, Rome, Italy.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maurizio Federico, Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161-Rome, Italy; e-mail: federico{at}iss.it.
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Z. Percario, E. Olivetta, G. Fiorucci, G. Mangino, S. Peretti, G. Romeo, E. Affabris, and M. Federico Human immunodeficiency virus type 1 (HIV-1) Nef activates STAT3 in primary human monocyte/macrophages through the release of soluble factors: involvement of Nef domains interacting with the cell endocytotic machinery J. Leukoc. Biol., November 1, 2003; 74(5): 821 - 832. [Abstract] [Full Text] [PDF]
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E. Olivetta, Z. Percario, G. Fiorucci, G. Mattia, I. Schiavoni, C. Dennis, J. Jager, M. Harris, G. Romeo, E. Affabris, et al. HIV-1 Nef Induces the Release of Inflammatory Factors from Human Monocyte/Macrophages: Involvement of Nef Endocytotic Signals and NF-{kappa}B Activation J. Immunol., February 15, 2003; 170(4): 1716 - 1727. [Abstract] [Full Text] [PDF]
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A. Varin, S. K. Manna, V. Quivy, A.-Z. Decrion, C. Van Lint, G. Herbein, and B. B. Aggarwal Exogenous Nef Protein Activates NF-kappa B, AP-1, and c-Jun N-Terminal Kinase and Stimulates HIV Transcription in Promonocytic Cells. ROLE IN AIDS PATHOGENESIS J. Biol. Chem., January 17, 2003; 278(4): 2219 - 2227. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
chain of
T-cell receptor leads to release of Fas-ligand from T cells in an
antigen-independent manner.
