Copyrightq1996, American Society for Microbiology
The Ras-Raf Pathway Is Activated in Human Immunodeficiency
Virus-Infected Monocytes and Participates in the Activation of NF-
k
B
LOLA FOLGUEIRA,1† ALICIA ALGECIRAS,2WILLIAM S. MACMORRAN,1
GARY D. BREN,1
ANDCARLOS V. PAYA1,2,3*
Divisions of Experimental Pathology1and Infectious Diseases,3Department of Immunology,2
Mayo Clinic, Rochester, Minnesota 55905
Received 28 July 1995/Accepted 21 December 1995
Persistent human immunodeficiency virus (HIV) infection of human monocytes and macrophages increases IkBadegradation, resulting in the activation of NF-kB, a key transcription factor in the regulation of the HIV long terminal repeat. The signal transduction pathways leading to NF-kB activation in cells of the monocytic lineage, especially those regulated by HIV infection, and their relevance in regulating viral persistence remain unknown. Both p21rasand its downstream Raf-1 kinase participate in the transduction of signals initiated from a variety of cell surface receptors and in the regulation of transcription factors. We have studied whether the Ras-Raf pathway is functional and participates in HIV-mediated NF-kB activation in monocytic cells. Con-stitutively active p21ras
(v-H-Ras) activates NF-kB-dependent transcription and induces the nuclear translo-cation of a bona fide p65/p50 heterodimer by targeting IkBa. In addition, the constitutively active form of Raf (Raf BXB) also increases the NF-kB-dependent transcriptional activity. Because of the similarity between HIV-and Ras-Raf-induced NF-kB activation in monocytic cells, we next tested whether HIV-induced NF-kB acti-vation was mediated by the Ras-Raf signal transduction pathway. Negative dominant forms of both Ras (Ras N17) and Raf (Raf 301) decreased the HIV- but not lipopolysaccharide-dependent NF-kB activation in U937 cells. Moreover, Raf-1 kinase activity was greater in HIV-infected than uninfected monocytic cells in in vitro kinase assays. Altogether, these results indicate that the Ras-Raf pathway is upregulated in HIV monocytic cells and participates in the virus-induced activation of NF-kB.
The Ras and Raf-1 proto-oncogene products serve as a central intermediates in many signaling pathways by connect-ing upstream tyrosine kinases with downstream serine/threo-nine kinases, such as a mitogen-activated protein kinase and mitogen-activated protein kinase (MKK or MEK) (reviewed in references 4, 7, 14, 19, 25, 35, 37, and 41). Ras is a membrane-localized guanine nucleotide-binding protein that is biologi-cally active in the GTP-bound state (8, 33). Raf is a serine/ threonine kinase located primarily in the cytosol (37, 41). Growth factors that stimulate cellular protein-tyrosine kinase activity enhance both the proportion of Ras bound to GTP and the kinase activity of Raf-1 (reviewed in references 30, 35, 37, and 41). The activation of Raf-1 in many cases is dependent on the activity of Ras, suggesting that Raf-1 functions downstream of Ras. The Ras-Raf pathway increases the transcriptional activities of different transcription factors, such as c-Jun, an important component of AP1 (6, 20, 44, 45, 51), and the
NF-kB/Rel family of transcription factors (16, 21).
NF-kB is a dimer of proteins, which in humans include p50, p52, p65, c-Rel, and Rel B, that share a highly conserved region, known as the Rel homology domain, which is respon-sible for DNA binding, dimerization, and nuclear location (re-viewed in references 3 and 50). Although different combina-tions of homo- and heterodimers exist, the typical form of NF-kB that is activated in response to extracellular signals is composed of a heterodimer of p50 and p65. NF-kB is localized in the cytoplasm, where it is complexed to inhibitor molecules
or IkBs (reviewed in references 3, 32, and 50). Of these, IkBa has been clearly demonstrated to participate in the regulation of NF-kB by different stimuli (3, 32, 50). Posttranslational modifications of IkBa, including phosphorylation and degra-dation, are both required to result in the release of NF-kB and its subsequent nuclear translocation (9–11).
Although macrophages have been identified as a main res-ervoir of persistent human immunodeficiency virus (HIV) rep-lication (22, 24), the molecular mechanisms that regulate viral persistence remain to be elucidated. The HIV regulatory pro-teins such as Tat and Rev require interaction with host cellular proteins for effective viral replication (reviewed in reference 23). HIV Tat requires not only the transactivation response region within the nascent RNA of HIV but also cis elements within the enhancer region of the long terminal repeat (LTR), including the transcription factor NF-kB (5, 28, 46, 49). NF-kB is a key regulatory molecule of transcription of multiple cellu-lar and viral genes, including the HIV LTR (3, 32, 38, 50). HIV infection of monocytic cells and macrophages results in in-creased nuclear translocation of NF-kB (2, 36, 40, 43, 48), at least by increasing the degradation of IkBa(36). The impact of this virus-host cell interaction in favoring viral persistence is unknown, although different studies indicate that interrupting NF-kB function results in decreased viral replication (27, 29, 42). To determine whether NF-kB activation by HIV plays a role in the regulation of HIV persistence in human monocytes and macrophages, we need to more fully understand the mech-anisms by which HIV activates NF-kB. Previous studies from our group have determined that the activation of this transcrip-tion factor by persistent HIV infectranscrip-tion involves kinases other than conventional protein kinase C isoenzymes (40), such as protein kinase C-z(21a). In addition, previous studies demon-strated that ras proto-oncogene product p21 activates the HIV LTR (1, 12, 47) via the enhancer region (1, 12). It is therefore * Corresponding author. Mailing address: Division of Experimental
Pathology, Mayo Clinic, 200 First St. SW, Guggenheim 501, Rochester, MN 55905. Phone: (507) 284-3747. Fax: (507) 284-3757. Electronic mail address: PAYA@MAYO.EDU.
† Present address: Department of Microbiology, Hospital 12 Oc-tubre, 28041 Madrid, Spain.
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possible that HIV infection of monocytic cells results in the activation of the Ras pathway, which, by regulating the func-tions of different transcription factors, could affect the upregu-lation of transcription of the HIV LTR and/or of cellular proteins other than p21rasthat may be required for optimal HIV replication in these host cells.
In this study, we have analyzed whether Ras and its down-stream target, Raf-1 kinase, are involved in NF-kB activation induced by HIV infection in monocytic cells. We demonstrate that both proto-oncogene products mediate activation of NF-kB in monocytic cells by targeting IkBa. In addition, by using dominant negative mutants of both Ras and Raf, we show that the Ras-Raf pathway is activated in HIV-infected monocytic cells and that it participates in HIV-induced NF-kB activation.
MATERIALS AND METHODS
Cells, HIV infection, and reagents.The human promonocytic U937 cell line was obtained from the American Type Culture Collection (Rockville, Md.), grown in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum (Intergen), and routinely tested for mycoplasma contamination. Ten million exponentially growing U937 cells were infected with the HIV LAVBRUstrain as previously described (36) and studied from days 25 to 50 postinfection. During this period, all cells are viable and over 80% express intracytoplasmic HIV p24 and contain the highest degree of HIV-induced NF-kB activity. At least four separate infections were used for these experiments. HIV infection was routinely monitored by detection of reverse transcriptase activity in the culture supernatants. Lipopolysaccharide (LPS) from Escherichia
coli O127:B8 was obtained from Difco (Detroit, Mich.) and stored in sterile
water at2208C.
Plasmids.The 3kB-con-luc plasmid contains three tandem copies of thekB motif of the HIV LTR cloned upstream of the minimal conalbumin-luciferase (con-luc) promoter reporter gene (36). The thymidine kinase–b-galactosidase (TK-bgal) plasmid is a mammalian reporter vector designed for expression of
b-galactopyranoside (b-Gal) in mammalian cells driven by the herpes simplex virus thymidine kinase minimal promoter (Clontech, Palo Alto, Calif.). cDNAs encoding IkBa(26) (obtained from Cetus Corporation), the constitutively acti-vated forms of Ras (v-H-Ras; obtained from Geoffrey Cooper, Harvard Univer-sity) (13) and Raf-1 (Raf BXB; obtained from T. Geppert, University of Texas— Dallas) (39), and the dominant negative mutants of Ras (Ras N17; obtained from Geoffrey Cooper) (13) and Raf (Raf 301; obtained from Michael Karin, Uni-versity of California, San Diego) (16) were cloned upstream the cytomegalovirus (CMV) expression vector pcDNA3 (Invitrogen, San Diego, Calif.).
Transfection assays.Plasmids were transfected in U937 cells either by the DEAE-dextran method or by electroporation. Briefly, 63106cells were incu-bated with 4mg of luciferase reporter plasmid, 8mg of control TK-bgal and 16
mg of plasmid pcDNA3-Ras N17 or pcDNA3-Raf 301 in a buffer containing 500
mg of DEAE-dextran (Pharmacia, Piscataway, N.J.) per ml for 20 min at room temperature; 23106transfected cells were harvested 24 h later, using cell culture lysis reagent (Promega, Madison, Wis.), andb-Gal activity was detected by using the Tropix Galacto-Light reporter assay (Tropix, Bedford, Mass.). The rest of the sample (43106) was harvested by the same procedure 16 to 20 h later, and luciferase levels were measured in a Berthold Lumat luminometer. For electroporation, 107cells per point were transfected with 2.5mg of luciferase reporter plasmid, 10mg of TK-bgal, and 2.5mg of plasmid pcDNA3-v-H-Ras, -Raf BXB, or -IkBa, using a Bethesda Research Laboratories Cell-Porator (800
mF and 300 V).b-Gal and luciferase activities were measured 8 and 24 h, respectively, after transfection. Total protein in an aliquot of each sample was measured by the Bradford technique (Bio-Rad) as previously described (36). Results are presented as luciferase units normalized tob-Gal units and are representative of at least three different sets of experiments.
Vaccinia virus infection.Molecular clones containing sequences encoding the constitutively activated form of p21ras(61L Ras), cellular p21ras(c-Ras), and the
dominant negative form of p21ras(Ras N17) were inserted by using the SmaI site
into the recombinant vaccinia virus vector pSC11 by methods described previ-ously (18) and provided by P. J. Leibson, Mayo Clinic. pSC11 alone was used as a control for vaccinia virus killing. Titration of the different vaccinia virus con-structs was performed as previously described (18). Titers of each stock ranged from 43108
to 53109
. Exponentially growing cells (107
) were infected with 5 PFU of the appropriate vaccinia virus per cell for 4 h, after which cells were harvested.
Nuclear extraction, gel mobility shift assay, and Western blotting (immuno-blotting).Cytosolic and nuclear fractions were extracted by using a modification of the method of Dignam et al. (17). For the electrophoretic mobility shift assay, 5mg of protein extract was incubated with [g-32
P]ATP-labeled double-stranded oligodeoxynucleotides corresponding to the tandem ofkB sequences of the HIV enhancer. The binding reaction was analyzed by electrophoresis in
nondenatur-ing 5% polyacrylamide gels. In DNA bindnondenatur-ing competition assays, unlabeled oligonucleotides were used in a 40-fold molar excess with respect to the corre-sponding radiolabeled probes. When indicated, 1ml of anti-NF-kB antibodies (anti-p65, anti-p50, anti-p52, and anti-c-Rel; Santa Cruz Biotechnology, Santa Cruz, Calif.) was added to supershift DNA-protein complexes.
Cytosolic proteins were extracted by the procedure used for nuclear extracts, separated by electrophoresis on sodium dodecyl sulfate (SDS)–10% polyacryl-amide gels, and transferred to an Immobilon-P membrane (Millipore, Bedford, Mass.). Immunoblotting was performed with commercially available anti-Ras and anti-Raf antibodies (Santa Cruz). A polyclonal anti-IkB-aserum was gen-erated by using a glutathione S-transferase (GST)–IkBa fusion protein (36). Immunoblotting was visualized by using an ECL (enhanced chemiluminescence) Western blotting detection kit (Amersham, Arlington Heights, Ill.).
Immunoprecipitation and in vitro Raf-1 kinase assays.HIV-infected and uninfected U937 cells (107
per point) were centrifuged and then resuspended in lysis buffer (20 mM Tris, 40 mM Na2P2O7, 50 mM NaF, 5 mM MgCl2, 10 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, 40 mMb -glycerophos-phate [pH 8.0], 40 mM 4-nitrophenyl phos-glycerophos-phate, 1 mM Na2VO4, 1 mM phenyl-methylsulfonyl fluoride, 20mg each of leupeptin, pepstatin A, and aprotinin per ml, 0.6mM okadaic acid, 0.5 mM phosphoserine, 0.5 mM phosphotyrosine, 1 mM phosphothreonine). Insoluble material was removed by centrifugation, and cell lysates were equalized for Raf-1 protein expression. SDS (0.1%) was added to the cleared lysates. Raf-1 proteins were immunoprecipitated with antibodies to Raf-1 (Santa Cruz), and the antigen-antibody complexes were collected with protein G-agarose beads (GIBCO-BRL, Gaithersburg, Md.). Immunoprecipi-tates were washed two times with lysis buffer (without okadaic acid or phos-phoamino acids) containing 0.1% SDS, once with washing buffer (50 mM Tris [pH 7.5], 5 mM octyl-b-glucopyranoside, 1 mM dithiothreitol, 40 mMb -glycer-ophosphate), and once with kinase buffer (40 mM Tris [pH 7.5], 80 mM NaCl, 8 mM MgCl2, 1 mM EGTA, 5 mM octyl-b-glucopyranoside). Kinase reactions were initiated with 20ml of kinase reaction mix (kinase buffer containing 2mg of kinase-deficient GST-MEK [provided by Robert Abraham, Mayo Clinic] and 10
mCi [g-32P]ATP per reaction). Samples were incubated at 308C for 20 min, and the reactions were terminated with 20ml of 43SDS-polyacrylamide gel electro-phoresis (PAGE) sample buffer (40 mM Tris-HCl [pH 8.0], 40% glycerol, 20% 2-mercaptoethanol, 4% SDS, 4 mM EDTA, 0.01% bromophenol blue). Solubi-lized proteins were fractionated by SDS-PAGE and electrophoretically trans-ferred to Immobilon-P, and phosphoproteins were visualized by autoradiogra-phy.
RESULTS The constitutively activated form of p21ras
, v-H-Ras, induces the translocation of the p65/p50 heterodimer of NF-kB in U937 cells.While the Ras-Raf pathway seems to be conserved in eukaryotic cells and studies with mouse fibroblastic cells indicate that v-H-Ras and Raf BXB are constitutively active, in human T-cell models, constitutively active Raf BXB is not functional in that it requires additional signals (39). Therefore, we first determined whether the constitutively activated forms of Ras and v-H-Ras could be sufficient to induce NF-k B-de-pendent transcription in human monocytic cells, using tran-sient transfection assays. Results shown in Fig. 1A indicate that cotransfection of an expression vector encoding the activated form of v-H-Ras (CMV-v-H-Ras) resulted in a sixfold induc-tion of gene expression of a 3 kB-cona-luc reporter gene in U937 cells cotransfected with the empty expression vector. This result suggests that the Ras pathway functions in human monocytic cells to mediate NF-kB activation.
To confirm these results and to gain further insight as to the mechanisms by which Ras activates NF-kB in monocytic cells, we used a recombinant vaccinia virus vector expressing the oncogenic form of p21ras, 61L. We first tested whether in-creased NF-kB DNA binding activity was present in nuclear extracts from U937 cells infected with a vaccinia virus vector expressing the constitutively activated form, Ras 61L. As shown in Fig. 1B, U937 cells infected with vaccinia virus ex-pressing Ras 61L (lane 6) and cells stimulated with LPS for 30 min (lane 2) contained increased NF-kB DNA binding activity compared with that present in nuclear extracts from U937 cells mock infected (lane 1) or infected with wild-type vaccinia virus (pSC11; lane 3) or vaccinia virus expressing cellular Ras (lane 4) or its negative dominant form, Ras N17 (lane 5).
We next studied the molecular composition of the
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tively active Ras-induced NF-kB complex by using competition with an excess of unlabeled NF-kB oligonucleotide and anti-bodies to the different Rel-related family members. As shown in Fig. 1B, the Ras-induced complex is composed of both p65 (lane 9) and p50 (lane 7) subunits but not p52 or c-Rel (lanes 8 and 10).
Lastly, we explored potential mechanisms by which the con-stitutively active form of p21rascould mediate NF-kB translo-cation. Having identified p65 as a component of the NF-kB complex, we investigated whether inhibitors of NF-kB that anchor p65 such as IkBaare modified in U937 cells infected with vaccinia virus vectors expressing the different Ras con-structs. The cytosolic extracts used for the experiment repre-sented in Fig. 1B were used for IkBa immunoblotting. As shown in Fig. 1C, the steady-state levels of IkBaare not mod-ified in U937 cells after a 4-h infection with the wild-type vaccinia virus, pSC11 (lane 2), or in those cytosols expressing the cellular c-Ras (lane 4) or Ras N17 (lane 5). However, infection with a vaccinia virus expressing the constitutively active form of p21ras(Ras 61L) resulted in decreased IkBa levels (lane 6), suggestive of increased degradation. Similarly, stimulation of LPS for 60 min caused a significant decrease of IkBa(lane 3) compared with untreated, uninfected cells (lane 1). The difference in the amount of NF-kB DNA binding activity in LPS-treated samples versus that induced by Ras 61L parallels the degree of IkBadegradation in the two samples. To control for homogeneous expression of the different forms of Ras by the vaccinia virus vectors in the cytosolic cell lysates from the experiments described above (Fig. 1B and C), an immunoblotting experiment using anti-p21ras antibodies was performed. As shown in Fig. 1C, increased p21raslevels were observed in cells infected with vaccinia virus constructs ex-pressing c-Ras, Ras N17, and Ras 61L (lanes 4 to 6). Alto-gether, these results demonstrate that p21rasis functional in an HIV-permissive monocytic cell line such as U937 and suggest that IkBais a target of this important second messenger.
IkBa selectively decreases Ras- and Raf-mediated NF-kB but not AP1 activation.We next explored whether the kinase downstream of p21ras, Raf-1, shown in other systems to medi-ate NF-kB activation (21), was also functional in U937 cells to activate NF-kB. For this, we used a constitutively activated form of Raf (Raf BXB) which was cotransfected with reporter genes into U937 cells. The constitutively active Raf-1 kinase (Raf BXB) induced a fivefold increase in the transcriptional activity of an NF-kB-dependent reporter gene (kB-luc) as com-pared with that induced by an empty expression vector (Fig. 2A). In addition, the induction of NF-kB-dependent transcrip-tional activity by the constitutively active form of Ras (v-H-Ras) was similar to that induced by Raf BXB. To confirm the specificity of NF-kB activation by the constitutively active forms of Raf and Ras, these effector plasmids were cotransfected with an expression vector of IkBa. Expression of IkBa selective-ly decreased the enhanced NF-kB activity mediated by both Raf BXB and v-H-Ras (Fig. 2A). Altogether, these results indicate that both Ras and Raf-1 kinase are functional in activating NF-kB-dependent transcriptional activity in the absence of other costimuli in U937, an effect which is reversible by IkBa. Because AP1 is a target of Ras and Raf in vivo (6, 44, 45), we tested whether AP1-dependent transcription was upregulated in U937 following expression of either the oncogenic Ras or constitutively activated Raf (Raf BXB) and, if so, whether such activation was inhibited by IkBa. As shown in Fig. 2B, v-H-Ras and to a lesser degree Raf BXB activated an AP1-luc concate-mer reporter gene. However, cotransfection of an IkBa ex-pression vector did not inhibit the Ras-Raf-induced AP1 ac-tivity but rather enhanced it, indicating that the inhibitory effect of IkBais specific to the Ras-Raf-induced activation of NF-kB.
p21ras
[image:3.612.66.291.71.472.2]and Raf-1 kinase participate in HIV-mediated NF-kB activation.Persistent HIV infection results in increased NF-kB DNA binding (p65/p50) and transcriptional activity in the nu-clei of monocytic cell lines and human macrophages (36). This
FIG. 1. The constitutively activated p21ras(v-H-Ras) activates NF-kB in
U937 cells. (A) U937 cells were electroporated with akB-luc reporter gene and pcDNA3 alone (CMV-) alone or a CMV promoter-enhancer driving the expres-sion of v-H-Ras (CMV-v-H-Ras). Results are the means of three separate ex-periments and are expressed as luciferase units normalized tob-Gal units. In these and in subsequent transfection experiments, the minimal promoter cona-luc was used as a control and shown not to be activated by the constitutively forms of Ras or Raf or by an extracellular stimulus (LPS). (B) Electrophoretic mobility shift assay of nuclear extracts from U937 cells uninfected (NI; lane 1) or infected with an empty vaccinia virus vector (pSC11; lane 3) or vaccinia virus vectors driving expression of the different forms of Ras (c-Ras [lane 4], Ras N17 [lane 5], and Ras 61L [lane 6]). Unstimulated and uninfected U937 cells were treated with LPS at 1mg/ml for 60 min (lane 2). Nuclear extracts from U937 cells infected with vaccinia virus expressing Ras 61L were further incubated with antibodies (Ab) against the different NF-kB members (p50 [lane 7], p52 [lane 8], p65 [lane 9], and c-Rel [lane 10]), or the DNA binding activity was competed for with a 40-fold modern excess of unlabeled NF-kB oligonucleotide (lane 11). The NF-kB complex is marked at the right. (C) Cytosols from the experiment de-scribed for panel B were separated by SDS-PAGE and immunoblotted with anti-IkBaor anti-p21ras
antibodies. The same amount of protein (20mg) was loaded in each lane. The experiments shown in panels B and C are representative of two additional experiments.
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model of virus-induced NF-kB activation in monocytic cells is shown in Fig. 3. NF-kB-dependent transcriptional activity and DNA binding activity are increased in chronically HIV-in-fected U937 cells (Fig. 3A; compare lanes 1 and 2); in this experiment, the increase was similar to that induced by LPS treatment of uninfected cells (lane 3). In addition, the in-creased DNA binding activity correlates with dein-creased steady-state levels of IkBa(Fig. 3B; compare lanes 1 and 3) similarly to that induced by LPS (lane 2). Lastly, we confirmed whether increased NF-kB DNA binding activity and decreased steady-state levels of IkBain HIV-infected U937 cells corre-lated with increased NF-kB-dependent transcriptional activity. As shown in Fig. 3C, a fourfold increase was observed in HIV-infected cells.
Because of the similarity between the Ras-dependent acti-vation of NF-kB (Fig. 1) and that of HIV (Fig. 3), we investi-gated whether Ras and Raf-1 are components of the signal transduction pathway initiated by HIV that leads to NF-kB activation. To test this hypothesis, the dominant negative forms of Ras and Raf-1, Ras N17 and Raf 301, respectively, were cotransfected with an NF-kB-dependent reporter gene into uninfected or HIV-infected U937 cells. The ras mutant decreased by 65% the HIV-induced NF-kB activation (Fig. 4A; compare lane bars 2 and 4). The basal NF-kB activity present in uninfected U937 cells was only partially inhibited (15% reduction) by Ras N17 (compare bars 1 and 3). Similarly, Raf 301 inhibited by 70% the NF-kB-dependent transcrip-tional activity present in HIV-infected U937 cells (Fig. 4B, bars 2 and 4) but not in uninfected cells (bars 1 and 3). Having demonstrated a selective effect of these dominant negative molecules on interfering with HIV-mediated NF-kB activa-tion, we wanted to ensure that this observation was not due to a selective increased expression of the CMV promoter-en-hancer region driving the expression of Ras N17 and Raf-301
in HIV-infected compared with uninfected U937 cells. Be-cause of the difficulty of demonstrating by immunoblotting detectable overexpression of either dominant negative mole-cule upon DEAE-dextran transfection, the CMV enhancer-promoter was cloned upstream of the luciferase reporter gene (CMV-luc) and cotransfected with the TK-bgal reporter gene into uninfected and HIV-infected U937 cells to evaluate CMV-driven expression. As shown in Fig. 4C, the activity of this enhancer-promoter construct was significant and present equally in both uninfected and HIV-infected cells, suggesting that the levels of expression of the dominant negative mole-cules of both Ras and Raf are similar in uninfected and HIV-infected cells.
[image:4.612.86.272.75.313.2]To establish the specificity of the Ras and Raf pathway involvement in the HIV-mediated NF-kB activation, we deter-mined whether other stimuli known to activate NF-kB in cells of the monocytic lineage such as LPS require the Ras-Raf
FIG. 2. IkBainhibits the induction of NF-kB but not of AP1 by v-H-Ras and of Raf BXB. U937 cells were cotransfected with akB-luc reporter gene, v-H-Ras
or Raf BXB driven by a CMV expression vector (■), and a CMV plasmid
[image:4.612.359.512.265.653.2]expressing IkBa(^). Normalization of transfection was as described for Fig. 1A. The results are the means of three separate experiments with the corresponding standard deviations.
FIG. 3. HIV-infected U937 cells contain increased NF-kB activity and de-creased IkBalevels. (A) Electrophoretic mobility shift assay of nuclear extracts from uninfected cells (NI; lane 1), HIV-infected cells (lane 2), or uninfected LPS (500 ng/ml)-stimulated cells (lane 3). The NF-kB complex is labeled at the left. (B) Immunoblot of IkBafrom cytosolic extracts from the experiment shown in panel A. The same amount of protein (30mg) was loaded in each lane. (C) NF-kB-dependent activity of akB-luc reporter gene in uninfected (NI) and HIV-infected (HIV) U937 cells. Normalization of transfection was as described for Fig. 1A.
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pathway. In experiments similar to those described above, an NF-kB-dependent reporter gene (kB-luc) was cotransfected with the CMV-Ras N17 or CMV-Raf 301, after which cells were stimulated with LPS. As shown in Fig. 5, the NF-kB activation induced by LPS was not blocked by the Ras and Raf mutants. Interestingly, the dominant negative form of Raf en-hanced the LPS-induced NF-kB activity. Thus using a multi-disciplinary approach, we have determined that Ras and Raf-1 proteins participate in the activation of NF-kB in HIV-infected monocytic cells.
Increased Raf-1 kinase activity in HIV-infected U937 cells.
Having demonstrated that the Ras-Raf pathway is functional in monocytic cells and that it participates in the activation of NF-kB induced by HIV, we speculated that this pathway must be upregulated by HIV infection. To test this, we focused on Raf-1 kinase activity. This kinase is the one that mediates and initiates subsequent activation steps following Ras activation; it has been shown to be a putative IkBa kinase (31) and thus
potentially results in the direct activation of NF-kB. To test the activity of Raf-1, in vitro kinase assays of immunoprecipitated Raf-1 from uninfected and HIV-infected cells were performed by using recombinant kinase-deficient GST-MEK as a sub-strate. Greater phosphorylation of GST-MEK was observed with immunoprecipitated Raf-1 from HIV-infected U937 cells than with Raf-1 from uninfected cells (Fig. 6). Although equal protein amounts of cytosolic lysate from uninfected and HIV-infected cells were used to immunoprecipitate Raf-1 in the two cell types, we confirmed that the increased Raf-1 kinase activ-ity present in HIV-infected cells was not due to increased immunoprecipitated Raf-1 by performing an immunoblot anal-ysis of the immunoprecipitate used in the kinase assay. In addition, no increased kinase activity was present in protein kinase C-zimmunoprecipitates from HIV-infected cells com-pared with uninfected cells when IkBawas used as a substrate (data not shown), indicating that only selective kinase activities (such as that of Raf-1) are present in infected cells. These results confirm that the Ras-Raf pathway is upregulated in HIV-infected U937 cells and therefore support the findings that this pathway mediates the NF-kB activation induced by HIV.
DISCUSSION
[image:5.612.107.248.71.423.2]Understanding how HIV modifies the physiology of a host cell is important to advance our knowledge of the pathogenesis of AIDS. In this regard, virus-host cell interactions that par-ticipate in the persistent replication of HIV within monocytes and macrophages need to be delineated. Previous studies from our group identified the activation of NF-kB as a specific virus-host cell interaction in these cells. Although it remains to
[image:5.612.352.514.74.160.2]FIG. 4. Ras N17 and Raf 301 selectively inhibit HIV-induced NF-kB activity. (A) Uninfected (NI) and HIV-infected (HIV) U937 cells were cotransfected with a 3kB-cona-luc reporter gene and the CMV expression vector alone or CMV-Ras N17 (Ras N17). No reduction of the basal activity of a cona-luc reporter gene was observed when it was cotransfected with the dominant nega-tive forms. Normalization of transfection was performed as for Fig. 1. Results are the means of three separate experiments; standard deviations are shown. (B) Same as panel A except Raf 301 was transfected instead of Ras N17. (C) Uninfected (s) and HIV-infected (o) U937 cells were cotransfected with a CMV-luc reporter gene and the TK-bgal reporter gene. Data are the means of two separate experiments.
FIG. 5. LPS activates NF-kB through Ras- and Raf-independent pathways in U937 cells. Uninfected U937 cells were cotransfected with a 3kB-luc reporter gene and the CMV expression vector alone (vector) or CMV expressing Ras N17 or Raf 301. Following transfection, cells were unstimulated (■) or stimulated with LPS (1mg/ml;u).
FIG. 6. In vitro kinase assay of Raf-1. Cell lysates from uninfected (NI; lane 1) and HIV-infected (HIV; lane 2) U937 cells were immunoprecipitated with an anti-Raf-1 antibody and incubated in an in vitro kinase assay using catalytically inactive GST-MEK as the substrate. GST-MEK phosphorylation in the absence of cell immunoprecipitate is shown in lane 3. The same immunoprecipitates were analyzed by immunoblotting with the anti-Raf-1 antibody. Preincubation of the anti-Raf-1 antibody with specific peptide (Santa Cruz) prior to tation did not result in the phosphorylation of MEK or in the immunoprecipi-tation of Raf-1 (data not shown). These experiments are representative of two additional experiments.
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[image:5.612.364.506.568.644.2]be definitively proven that this virus-induced activation directly participates in regulating viral persistence, accumulating data point to NF-kB as a key element not only in the regulation of viral reactivation from latency but also in maintaining effective persistent replication. Studies presented here have determined that a specific and relevant signal transduction pathway involv-ing p21rasand Raf-1 kinase is functional in monocytic cells and that it participates in the virus-induced activation of NF-kB.
p21ras has been identified in recent years as a key signal transduction molecule that mediates signals derived from cer-tain cell surface receptors to the nucleus and thus plays an important role in many cell functions (4, 7, 14, 19, 25, 35, 37, 41). Most of the work delineating the functions of Ras has been performed with murine fibroblasts and T-cell lines, and little is known as to whether it is functional in cells of the monocytic lineage. Because NF-kB has very recently been identified as a transcription factor which is activated by p21ras (12, 16, 21), we examined whether this activation occurred in monocytic cells, in which case it could provide a model to test whether HIV infection modifies this key second messenger and thus potentially mediates HIV-induced NF-kB activation. In addition, previous studies had identified Ras as an activator of the HIV LTR and suggested that such activation was mediated by the enhancer motifs binding NF-kB-like molecules (1, 12, 47). Using vaccinia virus vectors, we have shown that expres-sion of a constitutively active Ras molecule results in the nu-clear translocation of NF-kB and identified the p50/p65 het-erodimer as the main complex being targeted. While previous studies demonstrated that the transcriptional activity of
NF-kB-dependent reporter genes was activated by p21ras expres-sion (16, 21), it remained unknown through which mecha-nism(s) NF-kB was activated. It had been speculated that the Ras-mediated transcriptional activation of NF-kB-dependent promoters might be by modifying other transcription factors such as AP1, which while in a nonactivated state could inhibit NF-kB activation as a result of the physical association be-tween the two types of transcription factors (21). The advan-tage of transfection of vaccinia virus vectors over conventional transfection is the ability of the former approach to result in a larger population of cells that express the gene of interest. In this way, we have been able to identify the molecular complex of NF-kB and putative IkB molecules that are targeted by p21ras. Identifying IkBa as a target molecule of p21ras is of importance in that it will allow a better definition of the signal transduction pathways and steps present between p21rasand IkBa. Moreover, this experimental approach will enable us to determine the phosphorylation sites within IkBathat are pre-sumably activated by p21ras. The observation that oncogenic Ras expression and chronic HIV infection appear to target IkBain similar fashions led us to explore whether the Ras-Raf pathway was activated following HIV infection in monocytic cells. Studies in HIV-infected cells clearly correlated increased turnover of IkBain infected cells secondary to its enhanced proteolysis with decreased steady-state IkBa levels and in-creased NF-kB nuclear translocation (36). Also, the identifi-cation of IkBaas a target of HIV infection via upregulation of p21rasis of importance for understanding how HIV activates NF-kB and thus its role in regulating viral persistence and also in other steps of viral replication. Recent data have demon-strated that IkBamay regulate and inhibit the function of HIV
rev (52). It is therefore possible that decreased IkBalevels in
persistently HIV-infected cells have an impact at least at two levels: increase NF-kB function and allow for a more effective
rev function.
Identification of targets downstream of p21rasis a major aim in the signal transduction research field. Because of the
pleio-tropic functions of p21ras, it has been speculated that more than one target, and thus pathways, are located downstream of this G protein (20). To date, the best-characterized targets are members of the Raf kinase family, especially Raf-1. This ki-nase has been identified as an initiator of the mitogen-acti-vated protein kinase cascade, which targets a variety of tran-scription factors. More importantly, Raf-1 kinase has been linked to NF-kB activation and suggested to be an IkBakinase (31). Our results indicate that the constitutively active form of Raf-1, Raf BXB, is sufficient to result in the transcriptional activation of NF-kB-dependent reporter genes in monocytic cells, thus suggesting that p21rasmay mediate NF-kB activation in these cells via Raf-1 kinase. However, the main goal of our study was to determine whether p21rasand potentially Raf-1 kinase, if functional, were used during HIV infection to acti-vate NF-kB. Using a series of dominant negative molecules that interfere with the functions of these two second messen-gers, our results indicate that both p21ras and Raf-1 kinase mediate NF-kB activation in HIV-infected cells. In addition, the participation of Raf-1 kinase in HIV-infected cells was analyzed by determining its kinase activity, using a physiolog-ical substrate such as the Raf-1 downstream target, MEK. Such experiments documented a constitutively increased Raf-1 ki-nase activity in HIV-infected cells, demonstrating not only that specific extracellular stimuli can result in a transient activation of Raf-1 but also that persistent HIV infection of a host cell results in a constitutive activation of Raf-1. The steps that lie downstream of Raf-1 kinase to activate NF-kB in HIV-infected monocytic cells remain unknown. Future studies will need to assess whether MEK or its downstream kinase (ERK) partic-ipates in targeting IkBa or, alternatively, whether more re-cently identified pathways such as those targeting JNK or p38 (15) are involved. The fact that LPS activation of NF-kB was not abolished by the dominant negative form of Ras or Raf imply the presence of Ras-Raf-independent pathways that me-diate NF-kB in monocytic cells. This specificity of the use of second messengers by different stimuli to mediate the same effect is important. Interruption of p21rasand/or Raf would still allow physiological stimuli (i.e., LPS) but not HIV to activate NF-kB in these cells.
Although our studies establish a nexus between persistent HIV replication, NF-kB, and the Ras-Raf pathway, it is still unknown how HIV activates this pathway. p21rasbelongs to the G protein family, which is normally located in the cytosolic membrane, and has been shown to be activated by different ligand-receptor interactions by means of other second messen-gers such as the Src tyrosine kinase members (34). Future studies will need to focus on the upstream molecules that are targets of HIV infection to result in p21ras activation. Their identification will allow us to search for the viral protein(s) that mediate this unique and novel virus-host cell interaction. Lastly, while monocytic cell lines provide a good model with which to study signal transduction studies in HIV pathogenesis because of the possibility of generating large cell numbers and feasibility of gene transfection, future studies should address, at least indirectly, the functions and roles of specific second messengers in HIV-infected human macrophages. In summary, this study has provided new information that will advance our knowledge of the mechanisms by which the Ras-Raf pathway activates NF-kB and identified it as a starting point to search for upstream and downstream steps that could be targets of HIV infection to result in the activation of NF-kB.
ACKNOWLEDGMENTS
We thank Doug Hauschild for excellent secretarial assistance and the Paya laboratory for helpful discussion.
on November 9, 2019 by guest
http://jvi.asm.org/
L.F. is a recipient of a Mitsubishi Scholarship. C.V.P. is an American Foundation for AIDS Research scholar. This work was supported by NIH grant R01 AI36076-01.
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