HIV encodes several proteins, including Tat, that have been demonstrated to modulate the expression of receptors critical for innate immunity, including MHC class I, mannose receptor, and β2-microglobulin. We demonstrate that Tat targets the receptor tyrosine kinase recepteur d’origine nantais (RON), which negatively regulates inflammation and HIV transcription, for proteosome degradation. Tat decreases cell surface RON expression in HIV-infected monocytic cells, and Tat-mediated degradation of RON protein is blocked by inhibitors of proteosome activity. Tat specifically induced down-regulation of RON and not other cell surface receptors, such as the transferrin receptor, the receptor tyrosine kinase TrkA, or monocytic markers CD14 and ICAM-1. The Tat trans activation domain is required for RON degradation, and this down-regulation is dependent on the integrity of the kinase domain of RON receptor. We propose that Tat mediates degradation of RON through a ubiquitin-proteosome pathway, and suggest that by targeting signals that modulate inflammation, Tat creates a microenvironment that is optimal for HIV replication and progression of AIDS-associated diseases.
Macrophages, in addition to CD4+ T cells, are a major target for HIV and are potential long-term cellular reservoir for virus dissemination (1, 2). Furthermore, dysregulated proinflammatory activities of macrophages associated with HIV infection directly contribute to AIDS and the development of associated pathologies in the CNS, lung, lymph nodes, and skin (3, 4). Successful eradication of HIV infection will depend on a better understanding of the biochemical mechanisms that regulate HIV replication in macrophages.
HIV, like all viruses, has used a variety of strategies, such as down-regulation of immune receptors on the surface of cells, to avoid detection and elimination by immune responses (5). The HIV-encoded proteins gp120, Tat, and Nef have been shown to influence the expression of a variety of cell surface molecules (6). For example, the viral protein Tat has been demonstrated to modulate the expression of cell surface proteins such as MHC class I (7, 8), mannose receptor (9), and β2-microglobulin (10). Despite a large set of data regarding Tat functions, the exact molecular mechanism by which Tat affects cellular gene expression still remains elusive.
Tat is essential for efficient HIV replication and is required for provirus transcription initiation and elongation. Tat binds to the trans activation response element (TAR),4 which forms an RNA stem-loop structure present at the 5′-end of HIV transcripts (11, 12, 13, 14, 15, 16). In addition, Tat has been reported to have a variety of other activities not associated with its ability to regulate HIV transcription, including neurotoxicity (17), immunosuppression, and inhibition of Ag-induced lymphocyte activation (18, 19). Furthermore, Tat contributes directly to the dysregulation of cytokine expression inducing the production of IL-1, IL-6, TNF-α, TNF-β, TGF-β1, and IL-4R (20, 21, 22, 23, 24, 25). Tat has also been shown to regulate the expression of several molecules that could impact the immune response to HIV infection, including cell surface proteins such as MHC class I (7, 8), mannose receptor (9), HIV coreceptors CXCR4 and CCR5 (26, 27), MIP-1α (28), β2-microglobulin (10), cytokine receptors (29, 30), vascular endothelial growth factor (31), and integrins (32). The ability of Tat to modulate the expression of many genes with no TAR element implies that Tat acts by interacting with other cellular regulatory proteins (33). These functions of Tat contribute to viral persistence and dissemination by directly or indirectly modulating the host anti-HIV response.
The receptor tyrosine kinase recepteur d’origine nantais (RON), which belongs to the Met proto-oncogene family (34), is a critical regulator of macrophage function and inflammation (35). RON is expressed mostly on tissue resident macrophages and regulates multiple cellular responses, including proliferation, differentiation, apoptosis, phagocytosis, and cell motility (35, 36, 37). Macrophage-stimulating protein, the ligand for RON, is a potent inhibitor of proinflammatory mediators such as NO, IL-12, and TNF-α (38, 39, 40, 41), whereas it induces the expression of genes associated with wound healing and the resolution of inflammation, including scavenger receptor A, IL-1Rα, and arginase (42). Activated macrophages from RON knockout mice produce elevated levels of NO in vitro and in vivo, and have demonstrated increased susceptibility to endotoxic shock as well as to autoimmune diseases mediated by inflammation (43, 44, 45). RON knockout mice also have compromised cell-mediated immunity, as demonstrated by an increased susceptibility to Listeria monocytogenes infection (46). We have previously shown that RON inhibits HIV transcription (47, 48) and replication in macrophages, and that the expression of this receptor tyrosine kinase is decreased in brains of AIDS patients (47).
In this study, we demonstrate that Tat decreases cell surface RON expression by targeting this receptor tyrosine kinase for ubiquitin-mediated proteosome degradation. Considering the role of RON in regulating tissue resident macrophage activities and inflammation, these findings suggest an additional mechanism by which HIV escapes the immune system and creates a microenvironment that is favorable for virus replication and progression of AIDS.
Materials and Methods
The U937 promonocytic cell line (American Type Culture Collection) was cultured in RPMI 1640 medium supplemented with 5% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.2 M l-glutamine. The 293T human embryonic kidney cell line (American Type Culture Collection) was cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.2 M l-glutamine. U937-RON cells were generated by transducing a RON retroviral vector, murine stem cell virus (MSCV)-RON, as previously described (47). Peripheral blood macrophages were isolated from whole blood obtained from healthy HIV-1-seronegative donors. Mononuclear cells were obtained by differential centrifugation using a Ficoll/Histopaque (Sigma-Aldrich) gradient and adherence to plastic culture flasks, as previously described (49). Macrophages were separated from all blood cells by an initial adherence to plastic culture flasks overnight. After removing nonadherent cells, monocytes were cultured for 5–7 days to mature into monocyte-derived macrophages. Mouse peritoneal macrophages were obtained by peritoneal lavage with 10 ml of RPMI 1640 containing 10% FCS. Cells were incubated overnight and washed with PBS to eliminate all nonadherent cells. Cells were maintained at 37°C in a humidified incubator containing 5% CO2.
Plasmids, transient transfections, and luciferase activity assay
Pvuless-hRON was provided by R. Breathnach (Institut de Biologie, Nantes, France). MSCV-RON construct (47) and the RON mutant constructs were generated, as previously described (50). HXB.2, HIV-1 GST-Tat expression vectors (86R TK, GST-Tat 1 72R, GST-Tat 1 72R P18lS, and GST-Tat 1 48D TK) (51), Mtat30 (52), and Nef (53) were obtained from the AIDS Research Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The JRFL expression construct was provided by R. Connor (Dartmouth, NH) (54). DNA for transfections was prepared using plasmid purification systems from Marligen Biosciences following protocols provided by the manufacturer. Transfection of 293T cells was performed using TransIT-293 transfection reagent from Mirus. For transfection of U937 cells, DNA was added to 0.4 ml of U937 cells resuspended in serum-free RPMI 1640 medium. The cell suspensions were electroporated using a T820 square electroporation system (BTX), with 1 pulse for 20 mSec at 240 V in 4-mm cuvettes. Cells were resuspended in 5 ml of RPMI 1640 supplemented with 5% FCS. Transfection efficiency was assessed by cotransfecting pEGEP-N3 (BD Clontech) and monitoring enhanced GFP expression by fluorescence microscopy. Cell viability was confirmed by trypan blue staining. RON overexpression did not influence the transfection efficiencies of the different cell lines, as previously described (47). Chloramphenicol acetyltransferase (CAT) ELISA (Roche) was performed to determine the promoter activity of the RON gene in cells transfected with and without Tat DNA. Luciferase assay has been performed using a commercial luciferase assay kit (Promega) and a TD-20/20 luminometer (Turner BioSystems).
Generation of HIV infectious titers, infections, and transduction of primary macrophages
Replication-competent HXB.2 and replication-incompetent Mtat30 viruses were generated by cotransfecting 293T cells with 15 μg of cDNA for either HXB.2 or Mtat30, 3 μg of vesicular stomatitis virus-glycoprotein, and 3 μg of RSV-Rev by CaPO4 transfection (55). Transfection efficiency was assessed by measuring p24 levels (p24 ELISA; PerkinElmer). We consistently generated titers of 1.0 × 106 infectious particles/ml. Supernatants were collected and filtered with 0.45-μm syringe filter (Whatman). One milliliter of undiluted virus stocks was added to 1.0 × 106 cells for 24 h and then replaced with fresh medium. MSCV-RON retroviral construct was packaged in 293T cells by transfecting 15 μg of the viral DNA, 3 μg of vesicular stomatitis virus-glycoprotein envelope, 3 μg of pECO, and 3 μg of Tat by CaPO4 transfection. Supernatants were collected and filtered through a 0.45-μm syringe filter (Whatman). For some experiments, cells were treated with purified rHIV-1 Tat (obtained from the AIDS Research Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health) (56) for 24 h in 1% serum medium.
Proteosome activity was inhibited by treating the cells with the proteosome inhibitors MG-132 (Calbiochem) at a concentration of 5 μM at 37°C in 5% serum medium for 30 min and epoxomicin (Sigma-Aldrich) at a concentration of 1 μM at 37°C in 5% serum medium for 1 h. Lysosomal proteolysis pathway inhibitor ammonium chloride (NH4Cl; Sigma-Aldrich) was applied to cells at a concentration of 20 μM for a duration of 30 min in 5% serum medium. For inhibiting protein synthesis, cells were pretreated with 2 ng/ml cycloheximide (Sigma-Aldrich) in 5% serum medium for 1 h. For all experiments using inhibitors, cell viability was monitored by trypan blue staining.
RNA extraction and RT-PCR
Total cellular RNA was prepared using TRIzol reagents (Invitrogen), according to the manufacturer’s instructions. Tat expression was analyzed using Tat primers 5′-GGA ATT CAC CAT GGA GCC AGT AGA TCC T-3′ and 5′-CGG GAT CCC TAT TCC TTC GGG CCT GT-3′. The PCR program consists of the following steps: 50°C for 30 min, 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and 1 cycle of 72°C for 7 min. Murine RON mRNA was analyzed by RT-PCR using RON primers 5′-CAG CAG TGG ACA GCC TGT TCA-3′ and 5′-ATG CCT TCC ACT CGG AAG TGC-3′. The PCR program consists of the following steps: 94°C for 3 min, followed by 35 cycles of 93°C for 30 s, 60°C for 1 min, and at the end 72°C for 2 min.
Immunoprecipitation and immunoblots
Cells were washed twice with PBS, and protein extracts were prepared by treating cells with lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0 mM EDTA (pH 8.0), 2.0 mM sodium vanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1.0 mM PMSF, and 1.0 mM pepstatin) at 4°C for 30 min. Lysates were mixed with 2× SDS loading buffer containing DTT and heated at 100°C for 5 min before resolving by SDS-PAGE with 8% polyacrylamide. Proteins were transferred to nitrocellulose membrane (Pall) and blocked with 5% nonfat dry milk in PBS with 0.02% v/v Tween 20 (J. T. Baker Chemical). Rabbit anti-RON polyclonal Ab (Santa Cruz Biotechnology) was used to probe for RON. HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich) was used as the secondary Ab. For detection of β-actin, membranes were stripped with 100 mM 2-ME, 62.5 mM Tris-HCl (pH 6.7), and 2% SDS for 30 min at 55°C, and reprobed with mouse anti-β-actin Ab (Sigma-Aldrich) for 1 h at 25°C, which was detected with HRP-conjugated goat anti-mouse (Sigma-Aldrich) secondary Ab. Goat anti-CD71 Ab (Santa Cruz Biotechnology) was used to detect transferrin receptor expression. HRP-conjugated anti-goat IgG (Sigma-Aldrich) was used as the secondary Ab. TrkA was detected with anti-TrkA (Millipore) and the secondary anti-rabbit IgG conjugated with HRP conjugated (Sigma-Aldrich). MHC I Ab (Santa Cruz Biotechnology) was used to probe for MHC I expression. HRP-conjugated anti-rabbit IgG (Sigma-Aldrich) was used as the secondary Ab. Nef protein expression was detected with HIV-1 Nef antiserum (Shugars et al., 1993) and gp120 (JRFL) envelope protein with antiserum to HIV-1 gp120 obtained from National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health contributed by Chiron Corporation. Antiserum against RON was raised against the last 15 aa of the carboxyl tail. Anti-ubiquitin Ab (Pierce) was used to detect ubiquitinated form of proteins, and anti-rabbit IgG was used as the secondary Ab. Blots were developed using an ECL-plus kit (Amersham Biosciences) and exposed to films. For some blots, densitometry with a personal ImageQuant analysis tool (Amersham Biosciences) was performed to determine signal intensities.
For immunoprecipitation, cell lysates were precleared for 30 min with protein A/G (Santa Cruz Biotechnology). RON was immunoprecipitated with 1.0 μg of anti-RON Ab (Santa Cruz Biotechnology) and protein A/G agarose beads (Santa Cruz Biotechnology). Protein-bound beads were washed four times with PBS and resuspended in 1× SDS buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromphenol blue, 10% glycerol, and 100 mM DTT) and resolved by SDS-PAGE.
Cell surface protein biotinylation and ubiquitination
Cell surface protein biotinylation and isolation were performed using Pinpoint cell surface protein isolation kit from Pierce. U937-RON cells were labeled with EZ-Link sulfo-NHS-SS-biotin and then lysed and isolated with immobilized NeutrAvidin gel. The bound proteins were released by incubating with SDS-PAGE sample buffer containing 50 mM DTT. The flow-through and elution were kept for analysis. Western blot was performed using an anti-ubiquitin antisera (included with kit). Ubiquitination of RON receptor at the cell surface is performed by using ubiquitin enrichment kit from Pierce. Cell lysates were incubated with ubiquitin affinity resin overnight. Ubiquitinated forms of RON were eluted with SDS-PAGE loading buffer, and Western analysis was performed using anti-ubiquitin antisera mAb (included with the kit).
Tat decreases RON protein
We previously observed a correlation between HIV infection and decreased RON expression in brain tissue (47). One possible explanation for diminished RON expression is that HIV directly targets this receptor. Therefore, to address whether HIV down-regulates RON expression, U937-RON cells, a U937 monocytic cell line transduced with RON, were transfected with HIV HXB.2 cDNA plasmid, and RON expression was monitored by immunoblotting (Fig. 1 A). RON is expressed as a 160-kDa precursor protein and 140-kDa proteolytic product, although the detection of this doublet depends upon the resolution of the gel and the quality of the anti-RON Ab. We consistently observed a decrease in RON protein by >75% when cells were transfected with the HIV plasmid.
HIV encodes six accessory proteins that potentially influence cell function and HIV replication. In particular, Nef and Tat have been shown to have activities that alter gene expression, receptor turnover, cell growth, and differentiation (6). To determine which HIV protein(s) was potentially involved in down-regulating RON expression, 293T cells were cotransfected with expression vectors encoding RON and different HIV proteins. As shown in Fig. 1, B and C, Nef and the envelope protein gp120 did not affect RON expression, whereas transfected Tat decreased RON expression by 50 to 70%. Tat did not mediate a general decrease in protein expression, because no changes were observed in the expression of transferrin receptor in U937 or U937-RON cells transfected with Tat (Fig. 1,D). To determine whether Tat has the ability to overcome the suppressive effects of RON on HIV transcription, U937-RON and U937-MSCV (cells transduced with the empty vector) were cotransfected with Tat and a HIV long terminal repeat (LTR)-luciferase reporter (LTR-Luc) and assayed for luciferase activity. The presence of RON inhibited LTR activity by >2-fold compared with LTR activity in Tat-transfected U937-MSCV cell line. Moreover, Tat induces LTR activity by >2-fold in the presence of RON, suggesting that Tat is able to at least partly circumvent RON-dependent inhibition of HIV transcription (Fig. 1 E). The LTR-Luc reporter construct used in this experiment lacks the TAR element which is replaced by the luciferase gene; therefore, any Tat-induced transcription would reflect activities other than its ability to activate provirus transcription processivity.
Tat decreased the expression of RON in the context of both U937 monocytic cells and primary macrophages. For these experiments, cells were treated with rTat, which is rapidly taken up by cells, as an alternative to transfection (57, 58). rTat was capable of trans activating a transfected HIV-LTR-luciferase reporter plasmid, and did not induce cell death as determined by exclusion of trypan blue or propidium iodide exclusion and flow cytometry (data not shown). As shown in Fig. 2,A, rTat significantly decreased RON expression in U937-RON cells by >50% compared with cells that were not treated with Tat. The decrease in RON protein was rapid, occurring within 2 h upon addition of rTat to U937-RON cells and extending up to at least 24 h (data not shown). Similar results were observed in primary monocyte-derived macrophages transduced with RON following Tat treatment (Fig. 2,B). It is important to note that the addition of Tat did not alter the activity of the MSCV retroviral promoter used to express RON because no differences were observed in RON mRNA levels in cells transduced with RON retroviral vector cultured in the presence and absence of Tat (data not shown). In addition, the expression of GFP from a MSCV-enhanced GFP retroviral vector was not altered by the presence or absence of Tat (data not shown). The above experiments depend on overexpression of RON, in part because human primary tissue resident macrophages that express RON are not readily accessible; therefore, we tested whether Tat influenced the expression of RON in primary murine peritoneal macrophages. Addition of rTat to these cells diminished the expression of RON protein by >75% (Fig. 2,C). The ability of Tat to down-modulate RON in the context of primary mouse macrophages is independent of its ability to increase transcription processivity, because Tat is unable to interact with P-TEFb in the context of murine cells (59). Diminished RON expression was also observed following infection with HIV IIIB, whereas infection with HIV clone Mtat30, which expresses a missense Tat, did not alter RON protein levels (Fig. 2 D), indicating that the levels of Tat associated with an infection are sufficient to down-regulate RON.
Tat does not appear to regulate RON transcription. In cotransfection experiments, Tat was unable to trans activate the RON promoter (Fig. 3,A). Furthermore, treating primary mouse peritoneal macrophages with rTat did not alter RON mRNA levels (Fig. 3 B). Taken together, these data suggest that Tat is down-regulating RON independent of transcriptional regulation.
Tat increases ubiquitin-dependent proteosome degradation of RON
To gain insight into the mechanism by which Tat is targeting RON, we examined whether RON was being expressed at the cell surface in the absence and presence of Tat. Because available Abs to RON poorly detect cell surface staining, we used a strategy in which cell surface proteins were biotinylated and subsequently immunoprecipitated and purified using NeutrAvidin gel. In Tat-treated U937-RON cells, reduced biotinylated RON was present compared with U937-RON cells that were not treated with Tat, suggesting that Tat is inducing the internalization and subsequent degradation of the receptor (Fig. 4,A). There was no significant change in the expression of the surface receptor tyrosine kinase TrkA (Fig. 4,B) or the monocytic markers CD14 and CD54 (ICAM-1) on U937-RON cells in the presence and absence of Tat (Fig. 4, C and D), indicating that Tat is not leading to a general turnover of surface proteins. A modest decrease was observed for MHC I (Fig. 4 B) consistent with previous studies that have shown that Tat inhibits MHC I transcription (7, 8).
Previous studies have shown that Met and RON expression is regulated by proteosome-dependent degradation (60, 61, 62). To investigate whether Tat-mediated down-regulation of RON requires the proteosome pathway, we treated U937-RON cells with rTat protein in the presence and absence of the proteosome inhibitor MG-132. MG-132 blocked the ability of Tat to mediate RON turnover (Fig. 5,A). We observed similar results with epoxomicin, a selective proteosome inhibitor (data not shown). Ammonium chloride (NH4Cl), an inhibitor of the lysosomal proteolysis pathway in macrophages (63), or cycloheximide, a inhibitor of protein synthesis, had no effect on the RON degradation in the presence of Tat (Fig. 5, B and C).
Proteins are directed to the 26S proteosome for degradation by polyubiquitination, a posttranslational modification in which ubiquitin polypeptides are covalently linked to lysines on the targeted proteins. To determine whether Tat promotes RON polyubiquitination, 293T cells were cotransfected with RON and Tat plasmids, and subsequently, RON was immunoprecipitated and examined for polyubiquitination by immunoblotting with an anti-ubiquitin Ab. RON from the Tat-transfected cells resolved in a high m.w. smear, consistent with polyubiquitination, whereas less ubiquitinated RON was observed in cells that lack Tat (Fig. 5 D). Taken together, our data show that Tat-mediated down-regulation of RON occurs through a ubiquitin-proteosome pathway.
The Tat trans activation domain is required for degradation of RON
Tat protein is composed of five functional domains (Fig. 6,A) (64). The trans activation domain includes the N-terminal acidic region, the adjacent cysteine-rich domain, the core domain, and the basic domain, which are encoded by the first exon (1–72 aa) (51) and are able to fully activate the HIV-LTR (65). To determine which domain(s) of Tat is mediating the degradation of RON protein, we cotransfected various Tat mutants with a RON expression construct. The first 72 aa (Tat 72R) were sufficient to mediate down-regulation of RON, and disrupting the trans activation domain by either inserting a glycine and phenylalanine between aa residues 18 and 19 (72R P181S) or deleting aa 2–48 (48D TK) abolished the ability of Tat to regulate RON expression. Furthermore, the basic region, which mediates RNA binding and the Arg-Gly-Asp domain, was unable to alter RON expression (48D TK; Fig. 6,B). These data suggest that the trans activation domain of HIV Tat is required for regulating RON expression (Fig. 6 B).
Tat-mediated degradation requires the RON kinase domain, but not the docking sites
To investigate which tyrosine residues of RON are involved in Tat-mediated degradation of this receptor, we used a series of RON mutations in which critical tyrosines were mutated to phenylalanines (Fig. 7,A). Tyrosines 1353 and 1360 serve as a multifunctional docking site for several signaling molecules, including Gab-1, Grb2, PI3K, phospholipase C, and Src homology region 2 domain-containing phosphatase 1–2 (35). Mutating these residues individually or in combination (Y1353F, Y1360F, and Y1353/1360F) did not affect the ability of Tat to down-regulate RON protein. By contrast, the kinase-defective RON mutants, RON KD and RON Y1238/1239F, were not susceptible to Tat-dependent degradation of RON (Fig. 7 B), indicating that Tat-mediated RON turnover is dependent on the integrity of the kinase domain.
HIV-1 targets critical receptors that participate in the regulation of the immune response. For example, Nef has been shown to interact with several signal transduction proteins and down-regulates the cell surface expression of CD4, CD28, MHC class I molecules, and FcγRs (66, 67, 68). Tat has also been shown to repress the expression of multiple cell surface proteins such as MHC class I (7, 8), mannose receptor proteins (9), and β2-microglobulin (10). In this study, we demonstrate that Tat diminishes the expression of RON, a receptor tyrosine kinase that regulates inflammation and macrophage function, through a ubiquitin proteosome-dependent degradation.
Tat decreases RON expression in a variety of cell types, including primary murine peritoneal macrophages, indicating a lack of tissue and species specificity. However, the ability of Tat to target RON for degradation does not reflect a general turnover of cell surface receptors because TrkA, CD14, and ICAM-1 surface expression were not altered in presence and absence of Tat. Furthermore, RON expression was decreased by the addition of exogenous Tat. Although we do not know the exact mechanism by which rTat is mediating this activity, we have shown that the Arg-Gly-Asp domain, which is recognized by cell surface integrin receptors, is not required for modulating RON expression, whereas the trans activation domain is sufficient for this RON degradation. We suspect that rTat is rapidly entering the cells, even though the levels of Tat associated with an in vitro infection were sufficient to modulate RON expression. Tat has been reported to be released from infected cells and taken up by neighboring uninfected cells (57, 69), providing a mechanism by which HIV can compromise immune function of uninfected macrophages. Whether the levels of Tat present in the serum or tissue are sufficient to have an impact on cell function in vivo is controversial.
Tat targets RON for ubiquitin proteosome degradation, as demonstrated by polyubiquitination of RON in the presence of Tat and the observation that Tat-dependent degradation of RON was blocked with the proteosome inhibitors MG-132 and epoxomicin. This ability of Tat to down-regulate protein expression by ubiquitin-proteosome degradation is a novel function of Tat. A recent report also suggested that Tat-mediated proteosome-dependent degradation of microtubule-associated protein 2 accounted for the loss of microtubule-associated protein 2 and neuronal damage observed in the brain of AIDS patients with neurological dysfunctions (70). Tat has also been shown to interact with several subunits of the 26S proteosome and to modulate the 20S proteosome activity (71, 72). We have not observed a physical interaction between Tat and RON (data not shown), suggesting that Tat is targeting RON possibly by activating signal transduction pathways that negatively regulate RON. Consistent with this hypothesis is the observation that RON degradation requires a functional kinase domain. Previous studies have similarly shown that the degradation of the Met receptor tyrosine kinase requires kinase activity (73). RON and Met, as well as other receptor tyrosine kinases, have been shown to be degraded upon activation in part by the recruitment of the E3 ubiquitin ligase c-Cbl (60). The role of c-Cbl in Tat-dependent degradation of RON is currently being explored.
HIV Tat is a potent trans activator of transcription and is essential for viral replication (74, 75). This viral protein interacts with the TAR element, and in cooperation with host cellular factors, in particular P-TEFb, functions to activate provirus transcription (76, 77). Furthermore, Tat has been shown to activate NF-κB signaling to trans activate gene expression in the absence of TAR (78, 79, 80). Several reports have also shown that Tat represses the transcription of multiple genes such as MHC class I (7, 8), mannose receptor (9), and β2-microglobulin (10). However, the ability of Tat to regulate RON expression is independent of P-TEFb because Tat was able to induce RON degradation in mouse peritoneal macrophages, despite the inability of Tat to interact with the murine CycT1 subunit of P-TEFb. Exon 1 of Tat, which includes the trans activation domain, is required for down-modulation of RON, indicating that the trans activation domain most likely mediates protein-protein interactions that facilitate targeting of RON to the proteosome. It is unlikely that Tat is inducing the expression of additional genes that indirectly down-modulate RON because cycloheximide does not influence Tat-dependent degradation of RON.
RON signaling has been shown to repress the transcription of inducible NO synthase, TNF-α, and IL-12 (38, 39, 40, 41). We have previously demonstrated that RON is also able to inhibit HIV transcription (47, 48). Because HIV transcription is tightly linked to proinflammatory signals and RON signaling has been shown to inhibit these signals, decreasing RON expression would be advantageous for virus replication. In addition, decreased RON expression would lead to dysregulation of the cytokine network and compromised innate immunity, increasing the susceptibility to opportunistic infection and facilitating disease progression. We have previously shown that RON expression is repressed in brain samples of a subset of HIV patients (47). Although this decrease in RON expression may be a consequence of multiple mechanisms associated with chronic infection and inflammation, including changes in cell populations, we propose that HIV Tat, in part by down-regulating RON, contributes to a microenvironment that favors HIV replication and further facilitates tissue damage and disease progression.
We are grateful to Dr. Avery August for critical review of the manuscript and scientific discussions. Also, we thank Elaine Kunze and Susan F. Magargee at Pennsylvania State University flow cytometry core facility for valuable assistance, and Qingping Liu and Xin Wei for the RON mutant expression constructs. The following reagents were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: HIV-1 GST-Tat expression vectors from Dr. Andrew Rice; pMtat30 from Dr. Reza Sadaie; Nef expression vector from Drs. Yingying Li, Feng Gao, and Beatrice H. Hahn; HIV-1 Tat protein from Dr. John Brady; HIV-1 Nef antiserum from Dr. Ronald Swanstrom; and gp120 Ab from Chiron.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by funds from the Penn State Tobacco Formula Funds and National Institutes of Health Grant AI46261 (to A.J.H.), and American Heart Association Predoctoral Fellowship Grant 0415425U (to P.K.).
Abbreviations used in this paper: TAR, trans activation response element; LTR, long terminal repeat; RON, recepteur d’origine nantais; CAT, chloramphenicol acetyltransferase.