Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M302878200 on July 3, 2003

J. Biol. Chem., Vol. 278, Issue 37, 35812-35818, September 12, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/37/35812    most recent
M302878200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cook, J. A.
Right arrow Articles by Henderson, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cook, J. A.
Right arrow Articles by Henderson, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

CD28-dependent HIV-1 Transcription Is Associated with Vav, Rac, and NF-{kappa}B Activation*

Julie A. Cook {ddagger} §, Lee Albacker §, Avery August § ¶ and Andrew J. Henderson § ¶ ||

From the {ddagger}Graduate Program in Biochemistry, Microbiology, and Molecular Biology, the §Department of Biochemistry and Molecular Biology, and the Department of Veterinary Sciences, Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication, March 20, 2003 , and in revised form, June 27, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of HIV-1-infected T cells through the T cell receptor and costimulatory molecule CD28 induces proviral transcription; however, the mechanism behind this enhanced virus expression is unknown. Jurkat T cells and primary CD4+ T cells expressing a CD8{alpha}/CD28 chimeric receptor containing a mutation at tyrosine 200 in the cytoplasmic tail were unable to fully induce HIV-1 proviral transcription in response to CD8{alpha}/28 receptor cross-linking in comparison to CD28 costimulation. The loss of transactivation seen with the mutant chimeric receptor correlated with a decrease in Vav tyrosine phosphorylation. CD28-dependent activation of HIV-1 transcription also required the GTPase activity of Rac1, which was not activated during costimulation with the mutated receptor. Furthermore, the mutated receptor was unable to induce NF-{kappa}B DNA binding or transactivation, as demonstrated by electromobility shift assays and HIV-1 long terminal repeat and NF-{kappa}B-dependent reporter constructs. These studies show that signaling events initiated by tyrosine 200 of CD28 are required for efficient expression of HIV-1 transcription in activated T cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Engagement of the T cell receptor (TcR)1 and costimulatory molecule CD28 results in T cell proliferation, differentiation, and induction of cytokine expression, including IL-2. Human immunodeficiency virus type 1 (HIV-1) also utilizes T cell signaling events to ensure efficient virus transcription and regulation. Productive infection of CD4+ T cells by HIV-1 and efficient HIV-1 proviral transcription requires cell activation through the TcR and costimulatory molecules including CD28 (1–5). However, signaling through CD28 has been shown to inhibit HIV-1 infection by down-regulating chemokine receptor expression (6). This and other data suggests that CD28 serves as both a positive and negative regulator of HIV-1 infection and replication (1, 4, 5, 7–10). CD28 signaling is mediated by four tyrosine residues in its cytoplasmic tail, which when phosphorylated, recruit and activate various kinases, phosphatases, and adapter molecules including LCK, ITK, GRID, GRB2, MKP6, and PI3K leading to changes in IL-2 production as well as HIV-1 transcription (11–16). Deletion of all four tyrosine residues in the CD28 cytoplasmic tail results in a loss of its ability to enhance IL-2 production as well as HIV-1 transcription, demonstrating the necessity of these residues for proper signaling (17, 18). Tyrosine residues 188, 191, and 200 have been shown to positively regulate IL-2 production, but their effects on HIV-1 have not yet been examined (17). Tyrosine 173 has been shown to be involved in T cell activation, but how it influences IL-2 expression is not clear (19–24). We have shown that Tyr173, which recruits PI3K to the CD28 signaling complex, negatively regulates HIV-1 transcription by a Tat-dependent mechanism (18).

Signaling events initiated by the TcR and CD28 lead to increases in intracellular calcium, changes in cytoskeletal organization, and triggering of several kinase cascades. Activation of these signaling pathways targets various transcription factors including NF-{kappa}B, NFAT, AP-1, Sp1, and Ets-1 (25–28). NF-{kappa}B, NFAT, and AP-1, which have binding sites within the HIV-1 long terminal repeat (LTR), are induced by CD28 costimulation (26, 28–31). However, it is unclear which signals emanating from CD28 are responsible for the induction of these transcription factors and the activation of HIV-1 transcription.

One potential candidate is the guanine nucleotide exchange factor (GEF) Vav which has been shown to be targeted by CD28 signaling in the absence of TcR signaling (11, 15, 32, 33). Vav has multiple binding domains including SH2 and SH3 domains allowing it to serve as an adapter molecule for T cell signaling. It has also been suggested that Vav is essential for properly integrating TcR and CD28 signals (34). Negative regulation of Vav occurs through its interaction with cbl-b, which, upon CD28 ligation, releases Vav and is targeted for autoubiquitination and degradation (35). Previous studies have shown that downstream of CD28, Vav, in cooperation with Rac1, activates NF-{kappa}B (33, 36, 37).

Using Jurkat T cell lines and primary CD4+ T lymphocytes expressing chimeric CD8{alpha}/28 receptors with mutations in critical tyrosines located in the cytoplasmic tail, we have shown that CD28 signaling is required for efficient HIV-1 transcription (18). In this study, we specifically examine the role of Tyr200 in regulating HIV-1 transcription. We show that Tyr200 is necessary for efficient HIV-1 transcription in Jurkat T cells and primary CD4+ T cells through initiation of signaling events that enhance Vav phosphorylation, Rac1 activation, and NF-{kappa}B binding activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Primary Cells—Jurkat T cell line clone E6–1 obtained from ATCC were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.2 M L-glutamine, and 0.5% Fungizone. 293T human embryonic kidney cells and Chinese hamster ovary (CHO) cells expressing FcR{gamma}II receptors (CHO-Fc) (gift from Dr. I. Mellman, Yale University) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.2 M L-glutamine, and 0.5% Fungizone. Jurkat cell lines overexpressing CD8{alpha}/28 chimeric receptors 8WT and F200 were described previously (17, 18, 38). Expression of receptors was confirmed by flow cytometry. Primary blood lymphocytes were isolated from whole blood using a Ficoll/Histopaque (Sigma) gradient. Macrophages were separated by adherence to plastic overnight and CD4+ T cells were then positively selected from the non-adherent population using a CD4+ isolation kit (Dynal, Oslo, Norway).

Generation of HIV-1 Infectious Titers and Infections—Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 was generated by transfecting 293T cells with 15 µg of cDNA for either pNL4–3-Luc(+) Env(–) Nef(–) (HIV-luc) (39) or pHXBnPLAP(+)Nef(+) (HIV-PLAP) (40); obtained from NIH AIDS Research and Reference Reagent Program, NIH, 3 µg of RSV-Rev, and 3 µg LTR VSV-G (41) by CaPO4 transfection (42). 293T transfection efficiency was assessed by determining luciferase activity using a Promega luciferase kit (Madison, WI). Supernatants were collected and filtered through a 0.45-micron disc prior to infection. Typically, the multiplicity of infection of infectious supernatants was ~0.1–0.5. Cells were infected by culturing in the presence of virus stock for 12–24 h before replacing with fresh media. Cells were cultured for an additional 24 h before measuring luciferase activity to assess virus transcription. In some experiments, cells were infected with HIV-PLAP and 72-h post-infection HIV-PLAP positive cells were selected using CELLection Pan Mouse IgG kit (Dynal, Lake Success, NY) along with anti-human PLAP antibodies (Sigma).

Activation of T Cells—Jurkat cells were washed and serum-starved for 4 h prior to activation. CHO-Fc cells were plated at 2 x 105 cells/well in a 24-well plate, incubated for 12 h to allow adherence, treated with mitomycin-C (Sigma) at 10 µg/ml and incubated in the absence of serum for 2 h before using to activate the Jurkat cell lines. 1 x 106 Jurkat cells were activated by coculturing cells with the CHO-Fc and 0.1 µg/ml mouse anti-human CD3 and 1.0 µg/ml anti-human CD28 or 1.0 µg/ml anti-human CD8{alpha} antibodies (BD Pharmingen, San Diego, CA). Following stimulation, Jurkat cells were harvested and luciferase activity measured.

Transient Transfections—1.2 x 107 Jurkat cells were resuspended in 400 µl of RPMI 1640 and placed on ice for 15 min along with 15 µg HIV-luc, LTR-luc, –205LTR-luc, –158LTR-luc, or –205m{kappa}B-luc and then electroporated using a T280 square electroporation system (BTX, San Diego, CA) (39). For some experiments, 12 µg of LTR-luc and 10 µg of RacN17 (generously provided by Dr. M. A. Schwartz, The Scripps Research Institute, La Jolla, CA) or 10 µg of pEF empty vector DNA were added to cells. Cells were given 1 pulse for 25 ms at 240 V in a 4-mm cuvette. Cells were then cultured for 24 h and stimulated as described above. Primary CD4+ cells were stimulated for 12 h with 10 ng/ml PMA and 2 µg/ml PHA prior to electroporation. Following stimulation, 1.2 x 107 primary CD4+ T cells were resuspended in 400 µl of 10% fetal calf serum RPMI 1640 and placed on ice for 15 min with 20 µg of HIV-luc, and 20 µg of pMHneo 8WT or F200 (17). DNA was transfected into primary T cells by electroporation using the following conditions: 4 mm cuvette, 250 volts, 1 pulse, and 40 ms (T280 square electroporation system). Cells were cultured 24 h prior to stimulation by anti-CD3, anti-CD28, or anti-CD8{alpha}. Receptor expression was monitored by flow cytometry.

Immunoprecipitation and Immunoblots—Jurkat cells were either left uninfected or infected as described above with HIV-luc or HIV-PLAP. The infected Jurkat cells were serum-starved 4 h prior to stimulation with 0.1 µg/ml mouse anti-human CD3, 1.0 µg/ml anti-human CD28, or 1.0 µg/ml anti-human CD8{alpha} antibodies (BD Pharmingen) and cross-linked using 5.0 µg/ml goat anti-mouse Ig (New England Biolabs, Beverly, MA) for 5 min. Protein extracts were prepared with lysis buffer (10 mM Tris-CL (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 phenylmethylsufonyl fluoride, 1.0 mM pepstatin). Protein A/G (Santa Cruz Biotechnologies) was incubated with whole cell extracts for 2 h to preclear the lysates. Protein A/G beads were precoated with 1.0 µg of anti-Vav (New England Biolabs), washed twice in lysis buffer before adding to precleared whole cell extracts (5 x 106 cells/sample to 20 µl of beads) for 15 h at 4 °C. Protein-bound beads were washed three times in lysis buffer before adding 1x SDS loading buffer containing dithiothreitol. Samples were then treated at 100 °C for 5 min before resolving on 8% SDS-PAGE. Proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, Keene, NH) and associated Vav was detected with primary antibodies against phosphotyrosine or anti-human Vav (New England Biolabs). Blots were developed using the Amersham Biosciences ECL kit.

Transduction of Primary CD4+—Retroviral expression vectors for CD8{alpha}/28 8WT and CD8{alpha}/28 F200 were generated by blunt-ended ligation of a HindIII/EcoRI fragment containing the chimeric receptors from the original pMHneo vector into blunted EcoRI and XhoI sites of murine stem cell virus vector 2.2 (MSCV 2.2; generously provided by G. Nolan, Stanford University, CA). MSCV-8WT or MSCV-F200 retrovirus were then packaged in 293T cells by transfecting 15 µg of the viral DNA with 3 µg of VSV-G envelope, 3 µg of pECO, and 3 µg of Tat by CaPO4 transfection (42). Supernatants were collected and filtered through a 0.45-micron disc prior to infection. Typically, the multiplicity of infection of infectious supernatants was ~0.1–0.5. Cells were infected by culturing in the presence of virus stock and 10 ng/ml PMA and 2 µg/ml PHA (Sigma) for 12–24 h before replacing with fresh media. Cells were cultured an additional 3 days prior to sorting with CD8 positive isolation kit (Dynal, Oslow, Norway). Sorted cells were allowed to rest overnight prior to activation.

Rac GTP Exchange Assay—Cells were stimulated as described above for immunoprecipitations. Lysates were made according to Rac1 Activation Assay Kit (Upstate Technologies, Lake Placid, NY). Briefly, cells were lysed in magnesium lysis buffer (MLB) supplemented with 10 µg/ml leupeptin, 10 µg/ml apoprotin, 1 mM sodium vanadate, and 1 mM sodium flouride. Cell lysates were precleared for 10 min with GST beads. Lysates were then incubated with PAK-1 PBD-agarose for 1 h at 4 °C. Beads were washed three times in MLB and samples were prepared for electrophoresis by adding 1x SDS loading dye. Samples were boiled for 5 min and resolved by 12% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and GTP-bound Rac1 was identified by anti-Rac1 antibody.

Electrophoretic Mobility Shift Assays—Nuclear extracts from Jurkat and primary cells were prepared as previously described (43). Electrophoretic mobility shift assays (EMSAs) were performed by incubating 4 µg of protein from nuclear extracts with 4 µg of poly dIdC (Amersham Biosciences), 0.25 mM HEPES (pH 7.5), 0.6 M KCl, 9.0% glycerol, 1.0 mM EDTA, 7.5 mM dithiothreitol, 50 mM MgCl2. Reaction mixtures were preincubated with 50-fold excess specific or nonspecific competitors, or 0.5 µg of polyclonal antibodies against NF-{kappa}B subunits or C/EBP for 20 min at room temperature. Samples were loaded onto a 6% polyacrylamide gel and electrophoresed at 120 V in 0.5x Tris borate-EDTA. Probes for EMSA were generated by annealing oligonucleotides representing the HIV-1 NF-{kappa}B sites 5'-AGCTCCTGGAAAGTCCCCAGCGGAAAGTCCCTT-3' and 5'-AGCTAAGGGACTTTCCGCTGGGGACTTTCCAGG-3' and C/EBP site 5'-GATCGCCTAGCATTTCATCACACGT-3' and 5'-GATCACGTGTGATGAAATGCTAGGC-3'. Probes were generated by end filling with the Klenow fragment of Escherichia coli polymerase in the presence of [{alpha}-P32]dCTP (44).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Tyr200 Is Necessary for CD28-induced HIV-1 Transcription—We have previously shown that Tyr173 within the cytoplasmic tail of CD28 initiates a negative signal in CD28-mediated costimulation of HIV-1 transcription (18). However, CD28 signaling overall increases HIV-1 transcription suggesting that other residues must be required for this activity. We tested the role of Tyr200 in CD28-dependent HIV-1 transcription using CD8{alpha}/28 chimeric receptors in which the extracellular and transmembrane domains derived from CD8{alpha} were fused to the CD28 cytoplasmic domain, which is sufficient for T cell costimulation (17). Jurkat cell lines were generated that express CD8{alpha}/28 chimeric receptors, in which all four tyrosine residues are maintained (8WT), or the Tyr200 has been mutated to phenylalanine (F200) (17). CD8{alpha}/28 receptor expression was confirmed using antibodies directed against CD8{alpha} and quantified by flow cytometry. Expression of the 8WT or F200 chimeric receptor was comparable between both cell lines and did not affect CD3 or CD28 expression (Ref. 18, Fig. 1A). In order to determine if Tyr200 was necessary for provirus expression, 8WT and F200 cell lines were infected with an HIV-1 clone that included a luciferase reporter (HIV-luc) in place of the viral Nef gene (39). These cells were stimulated with different combinations of antibodies against CD3, CD8{alpha}, and CD28 and cross-linked with CHO-Fc as described under "Experimental Procedures." Luciferase activity was assessed as a measure of proviral transcription. Cells stimulated through the 8WT chimeric receptor plus CD3 showed induction of HIV-1 transcription comparable to that observed when they were stimulated through the endogenous CD28 plus CD3 (Ref. 18, Fig. 1B). However, stimulating cells through CD3 and the F200 receptor did not result in induction of proviral transcription over the levels of CD3 stimulation alone (Fig. 1B). Engagement of CD3, CD8{alpha}, or CD28 alone did not efficiently activate viral transcription. These results suggest that tyrosine residue 200 in CD28 is necessary for efficient induction of HIV-1 transcription following CD3 and CD28 activation.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1.
Tyr200 is required for CD28-dependent HIV-1 transcription. A, expression of CD8{alpha}/28 chimeric receptors in Jurkat cells. Cells were stained with FITC-conjugated isotype control (filled line), anti-CD8{alpha} (solid black line), or anti-CD28 (broken black line) and analyzed by flow cytometry. B, Jurkat cells stably transfected with either 8WT or F200 CD8{alpha}/28 chimeric receptors were infected with HIV-luc for 18 h. Cells were then stimulated on CHO-Fc cells with 0.1 µg/ml anti-CD3 and 1.0 µg/ml anti-CD8{alpha} or anti-CD28 for 12–18 h and assayed for luciferase activity. Data are from a single experiment performed in triplicate, which is representative of a total of three experiments. C, expression of CD8{alpha}/28 chimeric receptors in primary CD4+ T cells. Primary CD4+ T cells were isolated from whole blood and transiently transfected with 10 µg of HIV-luc and 20 µg of either pMHneo 8WT and pMHneo F200 and stained with FITC-conjugated isotype control (filled line), anti-CD8{alpha} 8WT (solid black line), or anti-CD8{alpha} F200 (broken black line) and analyzed by flow cytometry. Percentage of positive cells is noted in the legend. D, 24-h post-transfection, primary CD4+ T cells were stimulated on CHO-Fc cells with 0.1 µg/ml anti-CD3 and 1.0 µg/ml anti-CD8{alpha} or anti-CD28 for 12–18 h and assayed for luciferase activity. Data are the averages for three independent experiments performed in triplicate. Results were normalized to the anti-CD3 plus anti-CD28 endogenous response, which was considered to be 100%.

 

To determine whether these chimeric receptors were functional in primary T cells and mimic normal signals, 8WT and F200 receptors were transiently transfected with the HIV-luc cDNA into CD4+ T cells. Expression of the chimeric receptors was confirmed by flow cytometry with antibodies directed against CD8{alpha} (Fig. 1C). Receptors were stimulated with antibodies against CD3 plus CD28 or CD8{alpha} and cocultured with CHO-Fc for cross-linking. Activation through the wild type chimeric receptor resulted in induction of transcription that was comparable to that observed when endogenous CD28 was used as the costimulatory signal (Fig. 1D). However, cells transfected with the F200 mutant chimeric receptor and stimulated with anti-CD3 plus anti-CD8{alpha} were unable to induce HIV-1 transcription over background levels (Fig. 1D). These data show that the CD8{alpha}/28 chimeric receptors are functional in primary cells, as well as Jurkat cells, and suggest that Jurkat cells are a suitable model system for investigating the mechanisms of CD28 signaling. More importantly, results from both the primary CD4+ T cells and the Jurkat T cell lines demonstrate that Tyr200 is necessary for CD28-dependent HIV-1 activation.

Tyr200 Mediates Vav Phosphorylation and Rac1 Activity—To identify molecules downstream of Tyr200, changes in tyrosine phosphorylation were examined following stimulation with anti-CD3 plus anti-CD28 or anti-CD8{alpha}. A decrease in the phosphorylation of an ~98 kDa protein was consistently observed in cells activated through CD3 and F200 compared with those in which endogenous CD28 provided the costimulatory signal (data not shown). Vav is guanine nucleotide exchange factor (GEF) with a molecular weight of ~95 kDa that has been shown to be phosphorylated post-CD28 ligation (15, 36, 45). To determine if phosphorylation of Vav was altered in F200 cells, immunoprecipitations were performed using 8WT and F200 cells either untreated or receiving costimulation through the endogenous CD28 or chimeric receptor. Cells activated with anti-CD3 plus anti-CD28 resulted in an increase in Vav phosphorylation over unstimulated cells. Costimulation through the 8WT chimeric receptor resulted in an equivalent induction of Vav phosphorylation; whereas, cells receiving costimulation through the F200 chimeric receptor had significantly less phosphorylated Vav (Fig. 2). These data suggest that Vav is a critical downstream target of CD28 Tyr200 and plays a role in the activation of HIV-1 transcription.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2.
Tyr200 mediates Vav phosphorylation. 8WT or F200 cells were left untreated or stimulated with 0.1 µg/ml anti-CD3 and either 1.0 µg/ml anti-CD28 or 1.0 µg/ml anti-CD8{alpha} then cross-linked with 5 µg/ml goat anti-mouse (GAM) for 5 min. Protein A+G-agarose beads were used to pull-down Vav from whole cell lysates then probed for antiphosphotyrosine. Blots were reprobed with Vav specific antibodies. The blots shown are from two separate experiments, and the band intensities do not reflect differences in Vav levels in the individual cell lines.

 

Vav mediates guanine nucleotide exchange of Rac1 downstream of CD28 signals (36, 45, 46). To determine whether Rac1 was a critical signaling intermediate for CD28-dependent HIV-1 transcription, a Rac1 dominant negative mutant (RacN17) was transiently transfected along with the HIV-1 LTR linked to a luciferase reporter (LTR-luc) in the F200 cell line. Ectopic expression of RacN17 resulted in the inhibition of the endogenous CD3 plus CD28 signal to the level of induction of cells receiving costimulation through CD3 plus the F200 receptor (Fig. 3A). These data suggest that Vav and Rac1 are important in mediating responses downstream of CD28 Tyr200.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 3.
Rac1 is required for CD28 activation for HIV-1 transcription. A, F200 cells were transiently transfected with 12 µg of LTR-luc and 10 µg of dominant negative Rac1. Cells were allowed to recover for 18 h and then left unstimulated or stimulated with 0.1 µg/ml anti-CD3 plus 1.0 µg/ml anti-CD28 or anti-CD8{alpha} on CHO-Fc cells. These data are from a single transfection performed in triplicate that represents three independent experiments. B, 8WT or F200 cells were stimulated for 10 min with 0.1 µg/ml anti-CD3 plus 1.0 µg of anti-CD28 or anti-CD8{alpha} and cross-linked with 5 µg/ml goat anti-mouse antibody. Cell lysates were immunoprecipitated with PAK-1 PBD-agarose. Associated GTP-bound Rac1 was determined by immunoblotting with anti-Rac1 antibody. The blots shown are from two separate experiments, and the band intensities do not reflect differences in Rac1 levels in the individual cell lines.

 

To confirm that Rac1, a downstream target of Vav, is a target of signaling through Tyr200 we directly measured levels of activated GTP-bound Rac1. Cell lysates were prepared from 8WT and F200 cells following costimulation through CD28 or CD8{alpha} and a PAK PBD GST pull-down was performed to detect GTP-bound Rac1. Activation of the 8WT cells through CD3 and either CD28 or CD8{alpha} resulted in equivalent induction of GTP bound Rac1 over unstimulated cells (Fig. 3B). F200 cells stimulated through CD3 and CD28 resulted in an increase in GTP-bound Rac1, but costimulation through the F200 chimeric receptor did not result in any increase in GTP-bound Rac1 (Fig. 3B). These data support the conclusion that Tyr200, through the activation of Vav guanine nucleotide exchange activity, targets Rac1 to influence HIV-1 transcription.

CD28-dependent Activation of HIV-1 Transcription Requires NF-{kappa}B Induction—The inability of the mutant F200 receptor to induce HIV-1 transcription may reflect inappropriate activation of cellular transcription factors. The role of host transcription factors was examined by transient transfection with HIV-1 LTR luciferase reporter constructs (LTR-luc), which lack viral proteins that may potentially influence virus transcription (18). LTR transcriptional activity was increased 3.5-fold in response to anti-CD3 plus anti-CD28 stimulation compared with the unstimulated cells, whereas stimulation through CD3 and the F200 receptor did not result in significant induction of LTR activity (Fig. 4B), indicating that the Tyr200 residue of CD28 affects HIV-1 transcription by targeting cellular transcription factors.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4.
NF-{kappa}B binding sites are required for CD28-mediated HIV-1 transcription. A, schematic of the HIV-1 LTR highlighting canonical binding sequences for transcription factors and constructs used in these experiments. B, the Jurkat F200 cell line was transiently transfected with 10 µg of full-length or truncated LTR-luc constructs. Cells were left unstimulated or stimulated with 0.1 µg/ml anti-CD3 plus 1.0 µg anti-CD28 or anti-CD8{alpha} on CHO-Fc cells. Results were normalized to the unstimulated response, which was set at 1. Data are from one experiment performed in triplicate and represent one of three independent experiments.

 

To further explore the transcription factors that are downstream of Tyr200 signaling, a series of HIV-1 LTR-luc constructs harboring deletions and mutations were assayed in the F200 cell line for their ability to respond to CD28 signaling (Fig. 4, A and B) (39). Deleting ~342-bp upstream of the transcriptional start site did not alter LTR activity in response to CD3 and endogenous CD28 stimulation (Fig. 4B, –205 and –158). However, activation of F200 cells with anti-CD3 plus anti-CD8{alpha} did not induce transcription in any of the LTR-dependent reporters. These deletion constructs still contained the NF-{kappa}B and SP1 binding sites. Mutation of the NF-{kappa}B sites in the –205 LTR-luc abolished the ability of CD28 costimulation to induce HIV-1 transcription suggesting that NF-{kappa}B is required for full induction of HIV-LTR activity. The 8WT cell line was able to induce the LTR-, –205-, and –158-dependent reporters with both CD8{alpha} and CD28 stimulation but was not able to induce the mutant NF-{kappa}B reporter (data not shown).

To confirm that NF-{kappa}B transcriptional activity was deficient in the F200 cells, NF-{kappa}B, and AP-1 reporters linked to luciferase were transiently transfected into the parental, 8WT, and F200 Jurkat cell lines prior to T cell activation. As expected AP-1 and NF-{kappa}B-dependent reporters were induced by CD3 plus CD28 stimulation in the parental Jurkat cell line (Fig. 5A). The 8WT cells were able to activate transcription of both NF-{kappa}B- and AP-1-dependent promoters when stimulated through CD3 plus CD28 or CD8{alpha}. The F200 cell line was unable to efficiently activate NF-{kappa}B- and AP-1-dependent reporters following CD3 plus CD8{alpha} stimulation, consistent with Tyr200 being upstream of AP-1 and NF-{kappa}B (Fig. 5, B and C). The deletion of AP-1 sites did not decrease the ability of the –158 LTR reporter to respond to CD28 costimulation suggesting a minimal role for this factor in HIV-1 transcription. Therefore, we focused on the requirement of NF-{kappa}B in response to CD28 signaling.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5.
Tyr200 is required for activation of NF-{kappa}B and AP-1 downstream of CD28. A, Jurkat cells, B and C, 8WT and F200 cell lines were transiently transfected with 10 µg of AP-1-luc or NF-{kappa}B-luc and then stimulated with 0.1 µg/ml anti-CD3, 1.0 µg/ml anti-CD28, or 1.0 µg anti-CD8{alpha} as indicated on CHO-Fc cells. Cells were lysed 4–6-h post-stimulation and assayed for luciferase activity. Data are one representative experiment performed in triplicate and represents one of three independent experiments. Results were normalized to the endogenous anti-CD3 plus anti-CD28 response, which was set at 100%.

 

Further characterization of NF-{kappa}B activation was assessed by electrophoretic mobility shift assays (EMSA) performed with nuclear extracts from 8WT or F200 cells stimulated through CD3 and either endogenous CD28 or the chimeric CD8{alpha} receptor. In some experiments, cells were infected with HIV-PLAP, a recombinant HIV-1 clone that includes a placental alkaline phosphatase marker to positively select infected cells. This virus also encodes the viral protein Nef, which was replaced by the luciferase gene in the previous HIV-luc experiments. Nuclear extracts from HIV-1-infected 8WT and F200 cells stimulated with antibodies against CD3 plus CD28 resulted in an increase in complex formation compared with unstimulated cells, which showed little to no binding activity. 8WT cells stimulated through CD3 and CD8{alpha} resulted in similar levels of NF-{kappa}B binding when compared with endogenous CD28 costimulation (Fig. 6A). In F200 cells stimulated with CD3 plus CD8{alpha}, no significant increase in complex formation was observed (Fig. 6A). Complexes formed in response to CD3 plus CD28 stimulation were competed away upon the addition of excess unlabeled NF-{kappa}B probe, but not with an irrelevant unlabeled competitor. Furthermore, these complexes were supershifted with antibodies specific to NF-{kappa}B (data not shown). Identical EMSA patterns were observed with extracts from F200 cells that were not infected, indicating that viral proteins are not altering NF-{kappa}B binding activity post CD3 plus CD28 stimulation (data not shown).



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 6.
NF-{kappa}B DNA binding is compromised following costimulation through the F200 chimeric receptor. Nuclear extracts from 8WT and F200 cells infected with an HXB-PLAP Nef positive virus (A) or primary CD4+ T cells transduced with retroviruses encoding 8WT or F200 receptors (B) and sorted for receptor expression were unstimulated or stimulated with 0.1 µg/ml anti-CD3 and either 1.0 µg/ml anti-CD28 or anti-CD8{alpha} in the presence of CHO-Fc cells for 4 h. Extracts were incubated with 50 pg of 32P-labeled NF-{kappa}B probe in the presence or absence of 50-fold excess unlabeled NF-{kappa}B or C/EBP nonspecific competitor.

 

We also determined whether Tyr200 was necessary for NF-{kappa}B activation in primary CD4+ T cells by EMSA. CD4+ T cells isolated from whole blood were transduced with retroviral vectors carrying the 8WT and F200 chimeric receptors. Cells were sorted for CD8{alpha} expression and confirmed to be positive for chimeric receptors by flow cytometry. Populations from the sorted cells were ~90% positive for CD8{alpha} expression (data not shown). Sorted cells were stimulated with antibodies against CD3 plus CD28 or CD8{alpha} in the presence of CHO-Fc cells. 8WT cells induced similar NF-{kappa}B binding complexes following activation with anti-CD3 plus anti-CD28 or anti-CD8{alpha} (Fig. 6B). The F200 cells were able to induce NF-{kappa}B binding in response to CD3 plus CD28 signaling but no significant change in NF-{kappa}B binding activity was observed when costimulation was provided by the F200 chimeric receptor. The NF-{kappa}B binding observed in these cells was specific since unlabeled NF-{kappa}B probe but not unlabeled nonspecific probe competed for binding (Fig. 6B). These data confirm that NF-{kappa}B is downstream of Tyr200 and further implicates this pathway in regulating CD28-dependent HIV-1 transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we demonstrate that residue Tyr200 of the CD28 receptor is required for the activation of HIV-1 transcription in both Jurkat and primary CD4+ T cells. We also show that a dominant negative Rac1 inhibits LTR-luc responses and that mutation of Tyr200 decreases Vav tyrosine phosphorylation and Rac1 activation. These two signaling molecules have been shown to regulate NF-{kappa}B activation, which is in agreement with a loss of NF-{kappa}B reporter and binding activity in the F200 cell line. The decrease in NF-{kappa}B activity severely impairs HIV-1 transcription, consistent with previous studies indicating that this is a critical transcription factor for HIV-1 LTR activity and virus replication (26, 31, 47).

The CD28 receptor is an important costimulatory molecule for T cell activation and has four tyrosine residues and a proline-rich domain, which are involved in the recruitment and activation of downstream effector molecules (16). Studies examining the induction of IL-2 and HIV-1 transcription have shown that the activity and interplay of these tyrosine residues is complex and may act competitively or synergistically with one another (11, 17, 22, 23, 38, 48). Tyr173 has been identified as recruiting ITK, GRB-2, and PI3K to the receptor (11, 14, 15). We have previously reported that mutating Tyr173 or inhibiting PI3K activity increases HIV-1 transcription through a Tat-dependent mechanism (18). However, the contribution of Tyr200 in activating HIV-1 transcription does not require viral-encoded proteins such as Tat or Nef, but mediates changes in cellular transcription factors (18). We have not yet determined the roles of Tyr188 and Tyr191 in HIV-1 transcription. Taken together our results demonstrate that the Tyr173 and Tyr200 residues recruit different signaling complexes to activate distinct pathways leading to either negative or positive signals to the HIV-1 LTR.

CD28 activation leads to an increase in Vav phosphorylation, which has been suggested to be mediated by tyrosine Tyr173 (49); however, we show that Tyr200 also regulates Vav tyrosine phosphorylation. Vav has been shown to interact with Grb-2, SLP-76, ZAP-70, and Rac1, which contribute to the activation of NFAT and NF-{kappa}B (33, 36, 37, 45, 50). Furthermore, over expression of Vav along with SLP76 circumvented the need for CD3 and CD28 signals to activate NFAT reporters (32, 51). Similarly, overexpression of Vav plus Rac1 or MEKK1 were able to circumvent CD3 plus CD28 signaling to induce NF-{kappa}B reporter activity (36, 45, 46, 53). We confirmed that Rac1 is necessary for induction of the HIV-1 LTR and observe strong activation of the HIV-1 LTR when MEKK1 is constitutively active (data not shown). This would be consistent with reports that MEKK1 can activate NF-{kappa}B and may be involved downstream of Vav (36, 45, 46, 53). However, additional experiments are required to establish a direct role for MEKK1 in events downstream of CD28 Tyr200. Experiments using chemical inhibitors against p38 and MEK1 have suggested that these MAPK pathways are not involved in CD28 Tyr200 mediated HIV-1 transcription (data not shown).

The viral protein Nef has been shown to influence T cell activation through interactions with several signaling intermediates downstream of the TcR and CD28. Vav is a potential target of Nef, although how this interaction with Vav influences HIV-1 transcription has not been investigated (54). Furthermore, Nef potentially alters NFAT transactivation but, its role in regulating NF-{kappa}B transactivation is not well characterized (52). In this report, Nef expressing viruses were used to compare NF-{kappa}B activity downstream of CD3 plus CD28 and CD3 plus CD8{alpha} by gel shift analysis. Our data indicates that Nef does not compensate for the F200 defect as assayed by NF-{kappa}B binding, suggesting that Nef has little impact on CD28 signaling through Tyr200 and minimal affect on NF-{kappa}B-dependent HIV-1 transcription. Finally, viral proteins do not influence signaling from F200 as suggested by the LTR-reporter constructs, which do not encode the viral proteins.

These data taken together with other reports suggest that CD28 activation of Vav and Rac1 are critical steps in the activation of NF-{kappa}B (36, 37). Furthermore, we demonstrate that this pathway is necessary for the CD28-dependent HIV-1 transcription. Therefore, T cell induction of HIV-1 transcription in response to T cell activation is complex, requiring signals from both the TcR and CD28 to provide efficient transcription of the virus as shown in both the Jurkat T cells and primary CD4+ T lymphocytes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI46261 (to A. J. H.) and AI051626 [GenBank] (to A. A.). 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. Back

|| To whom correspondence should be addressed: Dept. of Veterinary Sciences, 115 Henning Bldg., Pennsylvania State University, University Park, PA 16802. Tel.: 814-863-0340; Fax: 814-863-6140; E-mail: ajh6{at}psu.edu.

1 The abbreviations used are: TcR, T cell receptor; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; CHO, Chinese hamster ovary; PLAP, placental alkaline phosphatase; Grb-2, growth receptor-bound protein 2; PI3K, phosphatidylinositol 3-kinase, NFAT, nuclear factor of activated T cells; AP-1, activator protein 1; SH2, Src-homology 2; SH3, Src homology 3; NF-{kappa}B, nuclear factor {kappa}B; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; EMSA, electrophoretic mobility shift assay; PBD, P21 binding domain. Back


   ACKNOWLEDGMENTS
 
We thank the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health for the recombinant virus clone pHXBn-PLAP-IRES-N+. RacN17 was generously provided by Dr. M. A. Schwartz (Scripps Research Institute). MSCV 2.2 was generously provided by Gary Nolan (Stanford). Also, we would like to thank Margaret Kensinger for generating the MSCV-8WT and MSCV-F200 retroviral constructs and Drs. Eileen Lee and Louis Hodgson for critical discussion and technical advice. Elaine Kunze, at Pennsylvania State University flow cytometry facility, provided excellent technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Reiley, J. L., Levine, B. L., Craighead, N., Francomano, T., Kim, D., Carroll, R. G., and June, C. H. (1998) J. Virol. 72, 8273–8280[Abstract/Free Full Text]
  2. Zack, J. A., Arrigo, S., Weitsman, S. R., Go, A. S., Haislip, A., and Chen, I. S. (1990) Cell 61, 213–222[CrossRef][Medline] [Order article via Infotrieve]
  3. Stevenson, M., Stanwick, T. L., Dempsey, M. P., and Lamonica, C. A. (1990) EMBO J. 9, 1551–1560[Medline] [Order article via Infotrieve]
  4. Levine, B., Mosca, J., Riley, J., Carroll, R., Vahey, M., Jagodzinski, L., Wagner, K., Mayers, D., Burke, D., Weislow, O., St. Louis, D. C., and June, C. (1996) Science 272, 1939–1943[Abstract]
  5. Carroll, R., Riley, J., Levine, B., Feng, Y., Kaushal, S., Ritchey, D., Bernstein, W., Weislow, O., Brown, C., Berger, E., June, C., and St. Louis, D. C. (1997) Science 276, 273–276[Abstract/Free Full Text]
  6. Secchiero, P., Zella, D., Curreli, S., Mirandola, P., Capitani, S., Gallo, R. C., and Zauli, G. (2000) J. Immunol. 164, 4018–4024[Abstract/Free Full Text]
  7. Barker, E., Bossart, K., and Levy, J. (1998) J. Immunol. 161, 6223–6227[Abstract/Free Full Text]
  8. Smithgall, M., Wong, J., Linsley, P., and Haffar, O. (1995) AIDS Res. Hum. Retroviruses 11, 885–892[Medline] [Order article via Infotrieve]
  9. Wong, J. G. P., Smithgall, M. D., and Haffar, O. K. (1997) Immunology 90, 281–285[CrossRef][Medline] [Order article via Infotrieve]
  10. Wong, J., Hezareh, M., Gunthard, H., Havlir, D., Ignacio, C., C. Spina, C. Spina, and Richman, D. (1997) Science 278, 1291–1295[Abstract/Free Full Text]
  11. Kim, H.-H., Tharayil, M., and Rudd, C. E. (1998) J. Biol. Chem. 273, 296–301[Abstract/Free Full Text]
  12. Ellis, J., Ashman, C., Burden, M., Kilpatrick, K., Morse, M., and Hamblin, P. A. (2000) J. Immunol. 164, 5805–5814[Abstract/Free Full Text]
  13. Marti, F., Krause, A., Post, N. H., Lyddanc, C., Dupont, B., Sadelain, M., and King, P. D. (2001) J. Immunol. 166, 197–206[Abstract/Free Full Text]
  14. August, A., and Dupont, B. (1994) Int. Immunol. 6, 769–774[Abstract/Free Full Text]
  15. August, A., Gibson, S., Kawakami, Y., Kawakami, T., Mills, G. B., and Dupont, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9347–9351[Abstract/Free Full Text]
  16. Ward, S. G. (1996) Biochem. J. 318, 361–377[Medline] [Order article via Infotrieve]
  17. Teng, J. M. C., King, P. D., Sadra, A., Liu, X., Han, A., Selvakumar, A., August, A., and Dupont, B. (1996) Tissue Antigens 48, 255–264[Medline] [Order article via Infotrieve]
  18. Cook, J. A., August, A., and Henderson, A. J. (2002) J. Immunol. 169, 254–260[Abstract/Free Full Text]
  19. Sadra, A., Cinek, T., Arellano, J. L., Shi, J., Truitt, K. E., and Imboden, J. B. (1999) J. Immunol. 162, 1966–1973[Abstract/Free Full Text]
  20. Crooks, M. E. C., Littman, D., Carter, R. H., Fearon, D. T., Weiss, A., and Stein, P. H. (1995) Mol. Cell Biol. 15, 6820–6828[Abstract]
  21. Hutchcroft, J. E., Franklin, D. P., Tsai, B., Harrison-Findik, D., Varticosvski, L., and Bierer, B. E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8808–8812[Abstract/Free Full Text]
  22. Lu, Y., Philips, C. A., and Trevillyan, J. M. (1995) Eur. J. Immunol. 1995, 533–537
  23. Ward, S. G., Wilson, A., Turner, L., Westwick, J., and Sansom, D. M. (1995) Eur. J. Immunol. 25, 526–532[Medline] [Order article via Infotrieve]
  24. Okkenhaug, K., Wu, L., Garza, K., Rose, J. L., Khoo, W., Odermatt, B., Mak, T., and Ohashi, P. (2001) Nat. Immunol. 2, 325–332[CrossRef][Medline] [Order article via Infotrieve]
  25. Lyakh, L., Ghosh, P., and Rice, N. R. (1997) Mol. Cell. Biol. 17, 2475–2484[Abstract]
  26. Li, Y., Mak, G., and B. Robert Franza, J. (1994) J. Biol. Chem. 269, 30616–30619[Abstract/Free Full Text]
  27. Rameil, P., Lecine, P., Ghysdael, J., Gouilleux, B., Kahn-Perles, B., and Imbert, J. (2000) Oncogene 19, 2086–2097[CrossRef][Medline] [Order article via Infotrieve]
  28. Kane, L. P., Lin, J., and Weiss, A. (2002) Trends Immunol. 23, 413–420[CrossRef][Medline] [Order article via Infotrieve]
  29. Sieweke, M. H., Tekotte, H., Jarosch, U., and Graf, T. (1998) EMBO J. 17, 1728–1739[CrossRef][Medline] [Order article via Infotrieve]
  30. Kinoshita, S., Su, L., Amano, M., Timmerman, L. A., Kaneshima, H., and Nolan, G. P. (1997) Immunity 6, 235–244[CrossRef][Medline] [Order article via Infotrieve]
  31. Bassuk, A. G., Anandappa, R. T., and Leiden, J. M. (1997) J. Virol. 71, 3563–3573[Abstract]
  32. Raab, M., Pfister, S., and Rudd, C. E. (2001) Immunity 15, 921–933[CrossRef][Medline] [Order article via Infotrieve]
  33. Michel, F., Mangino, G., Attal-Bonnefoy, G., Tuosto, L., Alcover, A., Roumier, A., Olive, D., and Acuto, O. (2000) J. Immunol. 165, 3820–3829[Abstract/Free Full Text]
  34. Turner, M., and Billadeau, D. D. (2002) Nat. Rev. 2, 476–486
  35. Bachmaier, K., Krawczyk, C., Kozieradzki, I., Kong, Y.-Y., Sasaki, T., Oliverira-dos-Santos, A., Mariathasan, S., Bouchard, D., Wakeham, A., Itie, A., Le, J., Ohashi, P. S., Sarosi, I., Nishina, H., Lipkowitz, S., and Penninger, J. M. (2000) Nature 403, 211–216[CrossRef][Medline] [Order article via Infotrieve]
  36. Marinari, B., Costanzo, A., Viola, A., Michel, F., Mangino, G., Acuto, O., Levrero, M., Piccolella, E., and Tuosto, L. (2002) Eur. J. Immunol. 32, 447–456[CrossRef][Medline] [Order article via Infotrieve]
  37. Dienz, O., Hehner, S. P., Droge, W., and Schmitz, M. L. (2000) J. Biol. Chem. 275, 24547–24551[Abstract/Free Full Text]
  38. King, P. D., Sadra, A., Teng, J. M. C., Liu, X., Han, A., Selvakumar, A., August, A., and Dupont, B. (1997) J. Immunol. 158, 580–590[Abstract]
  39. Henderson, A. J., Zou, X., and Calame, K. L. (1995) J. Virol. 69, 5337–5344[Abstract]
  40. Chen, B. K., Gandhi, R., and Baltimore, D. (1996) J. Virol. 70, 6044–6053[Abstract]
  41. Bartz, S. R., and Vodicka, M. A. (1997) Methods Enzymol. 12, 337–342
  42. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8392–8396[Abstract/Free Full Text]
  43. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Free Full Text]
  44. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  45. Salojin, K. V., Zhang, J., and Delovitch, T. L. (1999) J. Immunol. 163, 844–853[Abstract/Free Full Text]
  46. Kaga, S., Ragg, S., Rogers, K. A., and Ochi, A. (1998) J. Immunol. 160, 4182–4189[Abstract/Free Full Text]
  47. Calvert, I., Peng, Z., Kung, H., and Raziuddin. (1991) Gene (Amst.) 101, 171–176[CrossRef][Medline] [Order article via Infotrieve]
  48. Pages, F., Ragueneau, M., Klasen, S., Battifora, M., Couez, D., Sweet, R., Truneh, A., Ward, S. G., and Olive, D. (1996) J. Biol. Chem. 271, 9403–9409[Abstract/Free Full Text]
  49. Klasen, S., Pages, F., Peyron, J. F., A. Cantrell, D., and Olive, D. (1998) Int. Immunol. 10, 481–489[Abstract/Free Full Text]
  50. Raab, M., Da Silva, A., Findell, P. R., and Rudd, C. E. (1997) Immunity 6, 155–164[CrossRef][Medline] [Order article via Infotrieve]
  51. Herndon, T. M., Shan, X. C., Tsokos, G. C., and Wange, R. L. (2001) J. Immunol. 166, 5654–5664[Abstract/Free Full Text]
  52. Yoon, K., and Kim, S. (1999) J. Gen. Virol. 80, 2951–2956[Abstract/Free Full Text]
  53. Tao, L., Wadsworth, S., Mercer, J., Mueller, C., Lynn, K., Siekierka, J., and August, A. (2002) Biochem. J. 363, 175–182[CrossRef][Medline] [Order article via Infotrieve]
  54. Fackler, O. T., Luo, W., Geyer, M., Alberts, A. S., and Peterlin, B. M. (1999) Mol. Cell 3, 729–739[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
I. I. Knezevic, S. A. Predescu, R. F. Neamu, M. S. Gorovoy, N. M. Knezevic, C. Easington, A. B. Malik, and D. N. Predescu
Tiam1 and Rac1 Are Required for Platelet-activating Factor-induced Endothelial Junctional Disassembly and Increase in Vascular Permeability
J. Biol. Chem., February 20, 2009; 284(8): 5381 - 5394.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. B. Strasner, M. Natarajan, T. Doman, D. Key, A. August, and A. J. Henderson
The Src Kinase Lck Facilitates Assembly of HIV-1 at the Plasma Membrane
J. Immunol., September 1, 2008; 181(5): 3706 - 3713.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
Z. Zhang, A. Klatt, A. J. Henderson, and D. S. Gilmour
Transcription termination factor Pcf11 limits the processivity of Pol II on an HIV provirus to repress gene expression
Genes & Dev., July 1, 2007; 21(13): 1609 - 1614.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J.-E. Kim and F. M. White
Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in jurkat cells.
J. Immunol., March 1, 2006; 176(5): 2833 - 2843.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. E. Pontow, N. V. Heyden, S. Wei, and L. Ratner
Actin Cytoskeletal Reorganizations and Coreceptor-Mediated Activation of Rac during Human Immunodeficiency Virus-Induced Cell Fusion
J. Virol., July 1, 2004; 78(13): 7138 - 7147.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
O. Equils, Z. Madak, C. Liu, K. S. Michelsen, Y. Bulut, and D. Lu
Rac1 and Toll-IL-1 Receptor Domain-Containing Adapter Protein Mediate Toll-Like Receptor 4 Induction of HIV-Long Terminal Repeat
J. Immunol., June 15, 2004; 172(12): 7642 - 7646.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/37/35812    most recent
M302878200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cook, J. A.
Right arrow Articles by Henderson, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cook, J. A.
Right arrow Articles by Henderson, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement