The Adaptor Protein SLP-76 Regulates HIV-1 Release and Cell-to-Cell Transmission in T Cells

This information is current as Tirumuru Nagaraja, Appakkudal R. Anand, Helong Zhao of September 26, 2021. and Ramesh K. Ganju J Immunol 2012; 188:2769-2777; Prepublished online 8 February 2012; doi: 10.4049/jimmunol.1102106

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Adaptor Protein SLP-76 Regulates HIV-1 Release and Cell-to-Cell Transmission in T Cells

Tirumuru Nagaraja,1 Appakkudal R. Anand,1 Helong Zhao, and Ramesh K. Ganju

HIV-1 infection in T cells is regulated by TCR activation. However, the cellular proteins of the TCR pathway that regulate HIV-1 infection are poorly characterized. In this study, in HIV-1 infection, we observed a significant reduction of HIV-1 virus production in Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76)–deficient Jurkat T cells compared with wild-type and SLP-76–reconstituted Jurkat T cells. We further confirmed the role of SLP-76 in HIV-1 infection by small interfering RNA- mediated knockdown in MT4 cells and PBMCs. Structural-functional analysis revealed that the N-terminal domain of SLP-76 was important for regulating HIV-1 infection. Further mechanistic studies revealed that lack of SLP-76 impaired virus release, but did not affect viral entry, integration, and . We also showed that SLP-76 plays a critical role in cell-to-cell transmission of

HIV-1. Signaling studies revealed that SLP-76 associated with viral negative regulatory factor protein and multiple signaling Downloaded from molecules during HIV-1 infection. Furthermore, SLP-76 facilitated the association of negative regulatory factor and F-actin, suggesting that SLP-76 mediates the formation of a signaling complex that may regulate viral release via cytoskeletal changes. Taken together, our studies demonstrate a novel role for the adaptor molecule SLP-76 in regulating HIV-1 infection in T cells with the potential to develop innovative strategies against HIV-1. The Journal of Immunology, 2012, 188: 2769–2777.

cell receptor activation has been shown to modulate HIV-1 transduction downstream of several surface receptors including http://www.jimmunol.org/ replication in CD4+ T lymphocytes (1, 2). Quiescent TCR (11, 12). Furthermore, SLP-76–deficient (SLP-762/2) mice T T cells do not support efficient transcription, expression, demonstrate a complete block in thymic development in the ab- and integration of the HIV-1 genome (3, 4). TCR-mediated sig- sence of peripheral T cells (13, 14). Recent studies have also naling has been shown to regulate both pre- and postintegration demonstrated the importance of SLP-76 in actin cytoskeleton steps in the HIV-1 life cycle (1, 2). TCRs mediate signaling remodeling downstream of the TCR (15, 16). Actin cytoskeletal through various downstream signaling molecules, including the reorganization has been shown to play an important role in HIV-1 Src homology 2 (SH2) domain-containing leukocyte protein of 76 entry and release from the target cells (17–19). kDa (SLP-76). SLP-76 is a key adaptor protein that is indis- Several experimental pieces of evidence indicate that negative pensable for TCR activation and expressed in all hematopoietic regulatory factor (Nef), an HIV-1 protein required for efficient viral by guest on September 26, 2021 lineages except mature B cells (5). SLP-76 is a multifunctional replication in vivo and AIDS disease progression, plays an im- protein and mediates its functions through three modular domains: portant role in the modulation of TCR signaling (20–22). Nef is an an amino N-terminal acidic region with three tyrosine phosphor- ∼27-kDa myristoylated protein expressed soon after infection that ylation motifs, a central proline-rich domain, and a C-terminal associates with cell membranes and downmodulates an array of SH2 domain (6). The tyrosine-phosphorylated residues on the N cell-surface molecules including CD4, MHC class I /II, CD28, and terminus of SLP-76 have been shown to associate with the SH2 chemokine receptors (23, 24). In addition, Nef has been shown to domain of various proteins including Vav (7, 8), noncatalytic interact with various components of the TCR signaling pathway region of tyrosine kinase adaptor protein (Nck) (9), and IL-2– (20–22, 25, 26). Nef has also been shown to modulate actin cy- inducible kinase (ITK) (10). Biochemical and genetic evi- toskeletal remodeling and viral release (17, 19, 20, 25–28). dence has clearly established the importance of SLP-76 in signal In the current study, we have shown that SLP-76 plays an im- portant role in HIV-1 infection. Further elucidation of the molecular mechanism revealed that SLP-76 does not interfere with HIV-1 Department of Pathology, Ohio State University Medical Center, Columbus, OH binding, entry, and preintegrative steps, but mediates viral re- 43210; and Center for Retrovirus Research, Ohio State University Medical Center, lease and spread. Our studies indicate that SLP-76 associates with Columbus, OH 43210 1 Nef and multiple signaling molecules that lead to actin reorgani- T.N. and A.R.A. contributed equally to this work. zation, which in turn may regulate HIV-1 virus release from the Received for publication July 21, 2011. Accepted for publication January 6, 2012. cell. This work was supported in part by National Institutes of Health Grant 5R01HL087576 (to R.K.G.) and funds from the Ohio State University Medical Center. Materials and Methods Address correspondence and reprint requests to Dr. Ramesh K. Ganju, Department of Pathology, Ohio State University Medical Center, 1645 Neil Avenue, 185 Hamilton Hall, Columbus, OH 43210. E-mail address: [email protected] MT-4 cells, a human T cell line bearing human T cell virus type 1, Abbreviations used in this article: FL, full-length; ITK, IL-2–inducible T cell kinase; wild-type (WT) Jurkat cells (JE6.1 clone; National Institutes of Health MS, multiple-spliced; Nck, noncatalytic region of tyrosine kinase adaptor protein; AIDS Research and Reference Reagent program), SLP-76–deficient (SLP- Nef, negative regulatory factor; NT, nontargeting; PAK2, p21-activated kinase 2; 762/2) cell line (J14), and SLP-76–deficient cell line stably reconstituted qRT-PCR, quantitative RT-PCR; SH2, Src homology 2; siRNA, small interfering +/+ RNA; SLP-76, Src homology 2 domain-containing leukocyte protein of 76 kDa; with SLP-76 (J14/7611, SLP-76 ) (kindly provided by Gary Koretzky, SLP-762/2, Src homology 2 domain-containing leukocyte protein of 76 kDa-defi- University of Pennsylvania) were grown in RPMI 1640 medium (Medi- cient; VS, virological synapse; WT, wild-type. atech) supplemented with 10% (v/v) heat-inactivated FCS (Invitrogen), 100 U penicillin/ml, and 100 mg streptomycin/ml. Expression of SLP-76 in Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 reconstituted SLP-76 cells was maintained by using 500 mg G418/ml. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102106 2770 SLP-76 REGULATES HIV-1 INFECTION

PBMCs were isolated from heparinized venous blood collected from 37˚C to remove the uninternalized virus from the surface, and washed once healthy donors (American Red Cross, Columbus, OH). Because only cells with complete RPMI 1640 medium and five times with PBS. The cells are used and are without identifiers, this work is exempt from institutional were lysed using lysis buffer (1% Triton X-100), and viral entry was review board review. The blood was subjected to Ficoll-Paque density monitored by measuring the p24 levels in the cell lysates by ELISA. Total gradient centrifugation at 2200 rpm for 35 min. For the primary lymphocyte cell protein was calculated using Bradford assay, and all samples were culture, the cells were suspended in RPMI 1640 containing 10% FCS, normalized for protein content. 2 mM glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin, and PHA (5 mg/ml) for 3 d. Cells were then removed and placed in fresh medium Virus integration supplemented with recombinant human IL-2 (Advanced Biotechnologies, Host cell nuclear integration of HIV in T cell lines and PBMCs was analyzed Columbia, MD). The purity of the PBMCs was checked by flow cytometry by PCR as described previously (29). DNA was prepared by using a DNA using CD3 Ab. extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s Viral isolates protocol. DNA samples were used for the amplification of integrated DNA with Alu-LTR–specific primers (Table I) using the following cycling The HIV-1 CXCR4 tropic (HIV-1 IIIB strain) and CCR5 tropic isolates conditions: 95˚C for 10 min, then 40 cycles at 95˚C for 15 s, 60˚C for (HIV-1 BaL) used in this study were obtained from the National Institutes of 1 min, and 72˚C for 1 min. GAPDH was used as an internal control. Health AIDS Research and Reference Reagent Program. Determining proviral transcription of full-length and HIV-1 infection assay multiple-spliced HIV mRNA 2 2 WT, SLP-76 / , and SLP-76+/+ Jurkat cells were infected with HIV-1 IIIB RNA was prepared from 2 3 106 HIV-1 IIIB-infected WT and SLP-762/2 at 10 ng p24/106 cells. The cells were washed extensively after 2 h of cells using the QIAamp RNAeasy Mini Kit protocol (Qiagen). Full-length infection to remove the residual virus and incubated for different time (FL) and multiple-spliced (MS) HIV mRNA transcript levels were deter- points. In the case of small interfering RNA (siRNA) and mutant plasmid mined by a modification of a protocol described by Brussels et al. (30). Downloaded from transfected cells, the cells were infected at 20 ng p24/106 cells. HIV-1 p24 Quantitative RT-PCR (qRT-PCR) was performed using primers (200 nM) Ag levels in the culture supernatant were assessed with the HIV-1 p24 core specific for FL and for MS (Table I) using 400 ng sample RNA in a SYBR profile ELISA kit (Advanced Biosciences Laboratories). Abs Nef Ab and FITC-conjugated p24 Ab was obtained from Millipore (Bill- erica, MA). PE-conjugated CXCR4, allophycocyanin-conjugated CD4 Abs, http://www.jimmunol.org/ and isotype conjugate controls were obtained from BD Pharmingen (San Jose, CA). SLP-76, Vav1, NCK1, p21-activated kinase 2 (PAK2), and GAPDH Abs were obtained from Technology (Danvers, MA). HRP-conjugated anti–HIV-1 p24 Ab was obtained from Advanced Biosciences Laboratories. Alexa Fluor-conjugated secondary Abs and phalliodin-conjugated Alexa Fluor 568 was obtained from Invitrogen. Anti-FLAG Ab was obtained from Sigma-Aldrich (St. Louis, MO). Confocal microscopy

For the colocalization analysis of Nef and F-actin as well as Nef and SLP- by guest on September 26, 2021 76, cells were fixed and permeabilized using the Fix/Perm solution (BD Biosciences, San Jose, CA) for 20 min at 4˚C. The cells were washed three times with BD Perm/Wash buffer (BD Biosciences) and blocked with Normal Goat Serum (Invitrogen) for 1 h at 4˚C. The cells were washed with Perm/Wash buffer and incubated with respective primary Abs for 1 h at 4˚C. After the incubation, the cells were washed three times and incu- bated with respective Alexa Fluor conjugate (Alexa Fluor 488 or 568) Ab for 1 h at 4˚C. For the above confocal studies, the slides were mounted using Vectashield mounting medium with DAPI (Vector Laboratories) and then examined under an Olympus FV1000 confocal microscope (633/1.4 oil; Olympus). The pictures were analyzed using FV-10 ASW software. Flow cytometry For the analysis of surface expression of CXCR4 and CD4 receptors, cells were washed twice with PBS, resuspended in PE-conjugated CXCR4 and allophycocyanin-conjugated CD4 Abs or with respective isotype conjugates as a control (BD Pharmingen), and then incubated for 1 h at 4˚C. The cells were washed three times with ice-cold PBS and then analyzed by flow cytometry. To determine intracellular p24, cells were fixed and per- 2 2 meabilized using the Fix/Perm solution (BD Biosciences). The cells were FIGURE 1. HIV-1 virus production is abrogated in SLP-76 / Jurkat stained with anti-p24 mAb (KC57; Beckman Coulter) and analyzed by T cells. (A) The expression of SLP-76 in WT (JE6.1), SLP-762/2 (J14), flow cytometry using an FACSCalibur cytometer (BD Biosciences). The and SLP-76+/+ (J14/7611) Jurkat T cells was analyzed by Western blotting data were analyzed by CellQuest software (BD Biosciences). with anti–SLP-76 Ab (top panel). Anti-GAPDH Ab served as a control (bottom panel). (B) The surface expression of CD4 and CXCR4 recep- Virus binding and entry assay tors in the Jurkat clones was determined by staining the cells with allo- For virus binding assays, WT, SLP-762/2, and SLP-76+/+ cells (1 3 106 phycocyanin-conjugated CD4 Ab and PE-conjugated CXCR4 Ab or cells) were resuspended in complete RPMI 1640 medium, and the cells allophycocyanin- and PE-labeled isotype controls. Analysis of the recep- 6 were infected with HIV-1 IIIB at a concentration of 40 ng p24/10 cells. tors was performed by flow cytometry. The filled peaks represent Ab After 1 h incubation at 4˚C, the cells were extensively washed with ice- controls, and the open peaks indicate receptor expression. (C) HIV-1 IIIB cold PBS to remove unbound viruses, transferred to fresh tubes, and finally virus production in WT, SLP-762/2, and SLP-76+/+ Jurkat T cells was lysed in ice-cold lysis buffer (1% Triton X-100). Total cell protein was determined by analyzing HIV-1 p24 levels in the supernatants collected at estimated using Bradford assay, and all samples were normalized for protein content. Virus binding was monitored by measuring the amount of various time points postinfection by ELISA. Data are representative of p24 in the cell lysates by ELISA. For virus entry assay, the cells were three independent experiments. Virus production in the WT control: at day incubated with HIV-1 IIIB for 3 h at 37˚C. Following this incubation, the 4, 5.5 6 0.6; day 7, 18.9 6 2; day 9, 55.2 6 5; and day 14, 126 6 9 ng/ml cells were washed five times with PBS, treated with trypsin for 5 min at of p24 Ag. *p # 0.05 versus WT cells. The Journal of Immunology 2771

Green assay system using a Realplex Cycler (Eppendorf, Westbury, NY). Western blot analysis PCR conditions included 95˚C for 3 min (1 cycle), 94˚C for 1 min, 56˚C for 1 min, and 72˚C for 1 min (40 cycles), followed by 4˚C for 10 min. Equivalent amounts of protein extracts were run on a 4–12% gradient acrylamide gel (NuPAGE Bis-Tris gel; Invitrogen) and transferred onto Cell–cell transmission nitrocellulose membranes. Immunodetection involved specific primary Abs, appropriate secondary Abs conjugated to HRP, and the ECL Western The cell-to-cell transmission assay was carried out as previously described blotting detection system (GE Healthcare). (31), with some modifications. Briefly, WT and SLP-762/2 cells were infected with HIV-1 IIIB at 20 ng/106 cells for 3 d. After 3 d of infection, Statistical analysis when the cells showed 60–75% HIV-1 p24 positivity, these donor cells 6 were counted and cocultured with target TZM-bl cells at different ratios Reported data are the means SD of at least three independent experi- (donor/target ratios: 1:1; 1:5, and 1:10). After 24 h, the donor cells were ments performed in duplicate or triplicate. The statistical significance was removed by aspiration of the medium containing the Jurkat cell clones. determined by the Student t test. The TZM-bl cells were then incubated with fresh medium for 3 d, after which they were lysed. An equal amount of protein was used for luciferase Results activity using the Luciferase Reporter Kit (Promega). Efficiency of cell– 2/2 cell transmission was measured in terms of relative luciferase units. Differential susceptibility of the SLP76 and WT T cell clones to HIV-1 infection Transfections We first determined the role of SLP-76 in HIV-1 infection using To analyze the SLP-76 domains important for the HIV-1 infection, we SLP-762/2, WT, and SLP-76+/+ Jurkat cell lines. The SLP-762/2 transfected the respective domain mutated plasmids (D1–156, D224–244, and R448K) into SLP-762/2 cells using the Nucleofection Kit V (Program Jurkat cell variant expressed minimal amounts of SLP-76 in 2/2 +/+ S018; Amaxa Biosystems). WT and SLP-76 cells transfected with comparison with the WT and the SLP76 Jurkat cells (Fig. 1A). Downloaded from control plasmid were used as controls. After 24 h transfection, equal We also analyzed the expression of HIV-1 receptor, CD4, and numbers of cells were used for the HIV-1 infection. The expression of coreceptor CXCR4 in these cells. The SLP762/2 cells expressed different fragments in Jurkat cells was analyzed after 48 h transfection by CXCR4 and CD4 at levels similar to those observed in the WT and Western blot. SLP-76–mutated plasmids were kindly provided by Gary +/+ Koretzky (University of Pennsylvania). SLP-76 Jurkat cells (Fig. 1B). We then studied the role of SLP- 76 in HIV-1 virus production by infecting the WT and SLP-762/2 Small interfering RNA-mediated knockdown Jurkat cells with the laboratory-adapted X4-tropic virus HIV-1 http://www.jimmunol.org/ siRNA-mediated knockdown of SLP-76 was performed using ON- IIIB. The supernatant was collected at various time points and TARGETplus SMARTpool SLP-76 siRNA (Dharmacon, Boulder, CO), analyzed for p24 Ag levels by ELISA. The SLP-762/2 Jurkat cells according to the manufacturer’s protocol. Briefly, MT4 cells or PBMCs showed reduced HIV-1 virus production in comparison with the were transfected with 100 or 200 nM siRNA using the Amaxa system control WT Jurkat cells (Fig. 1C). We observed an increasing (Amaxa Biosystems), as mentioned above. The respective, Non-Targeting 2/2 siRNA SMARTpool was used as a control. SLP-76–mediated knockdown reduction in HIV-1 virus production in SLP-76 cells over was estimated by Western blot analysis after 48 h of the initial transfection. various time periods. To determine whether the observed differ-

FIGURE 2. Knockdown of SLP-76 in MT4 cells and by guest on September 26, 2021 PBMCs leads to reduced HIV-1 virus production. (A) MT4 cells were transfected with SLP-76–specific siRNA and NT siRNA using nucleofection (Amaxa Biosystems). The knockdown of SLP-76 expression was analyzed by Western blot analysis with anti–SLP- 76 Abs (top left panel). Anti-GAPDH Ab served as a control (bottom left panel). The siRNA-transfected cells were infected with HIV-1 IIIB 24 h after trans- fection. After 48 h, HIV-1 virus production in these cells was determined by measuring HIV-1 p24 levels in the supernatant by ELISA (right panel). Virus pro- duction in NT siRNA-transfected cells was 6.2 6 0.8 ng/ml p24 Ag. (B) PHA-stimulated PBMCs were transiently transfected with NT siRNA or SLP-76– specific siRNA for 48 h. The knockdown of SLP-76 expression was analyzed by Western blot analysis with anti–SLP-76 Abs (top panel). Anti-GAPDH Ab served as a control (bottom panel). (C) After transfection of PHA-stimulated PBMCs with respective siRNAs, the cells were analyzed for the viability by trypan blue assay (left panel), and the activation of the cells was analyzed by flow cytometry using CD38 Ab (right panel). (D) Twenty-four hours after transfection, cells were infected with either X4 tropic HIV-1 IIIB virus (left panel) or R5 tropic HIV-1 BaL virus (right panel) virus. Forty-eight hours later, the supernatants were collected and HIV-1 p24 levels determined by ELISA. Data are representative of three independent experi- ments. Virus production in NT siRNA-transfected cells was 511 6 36 (in case of X4 tropic virus infection) and 260 6 40 pg/ml of p24 Ag (for R5 tropic virus in- fection). *p # 0.05 versus NT siRNA-transfected cells. 2772 SLP-76 REGULATES HIV-1 INFECTION ence in HIV-1 virus production between the WT and SLP-762/2 cells was due to the lack of SLP-76 molecule, HIV-1 virus pro- duction was evaluated in SLP-762/2 cells that were reconstituted with normal human SLP-76 (SLP-76+/+). Susceptibility to HIV-1 infection in the SLP-76+/+ Jurkat cells was restored to the levels observed in the WT Jurkat cells (Fig. 1C), indicating that SLP-76 is important for HIV-1 virus production. Knockdown of SLP-76 in T cells leads to a decrease in HIV-1 virus production To further confirm by an independent method the effect of SLP-76 downregulation on HIV-1 virus production in T cell lines and pri- mary cells, we used siRNA-driven SLP-76 knockdown in MT4 cells and PBMCs. As shown in Fig. 2A, left panel, there was significant knockdown of SLP-76 in MT4 cells transfected with the SLP-76 siRNA compared with the nontargeting (NT) siRNA. When these transfected cells were infected with HIV-1 IIIB (Fig. 2A, right panel), there was ∼50% reduction in virus production in SLP-76 siRNA-transfected cells in comparison with NT siRNA-transfected Downloaded from cells, thus corroborating the results observed with the SLP-762/2 Jurkat T cell line. Furthermore, we also analyzed the effect of SLP- 76 in PBMCs by siRNA-mediated downregulation of SLP-76. We observed significant knockdown of SLP-76 in PBMCs transfected with SLP-76 siRNA compared with the NT control (Fig. 2B). There

was no difference in viability between NT siRNA and SLP-76 http://www.jimmunol.org/ siRNA-transfected PBMCs as demonstrated by trypan blue stain- FIGURE 3. The amino-terminal domain of SLP-76 is essential for A ing (Fig. 2C, left panel). We also determined that there was no HIV-1 virus production. ( ) Schematic representation of the WT and B difference in activation status of the PHA-stimulated PBMCs upon mutant SLP-76 constructs. ( ) The SLP-76 WT and mutant plasmids were transiently transfected into SLP-762/2 cells using nucleofection (Amaxa siRNA transfection, using CD38 as a marker. (Fig. 2C, right panel). Biosystems). The transfected cells were lysed after 48 h and analyzed by When the SLP-76 siRNA-transfected PBMCs were infected with Western blot analysis with anti–SLP-76 Ab. (C) The transfected cells and HIV-1 IIIB (X4 virus), the cells showed reduced virus production in control cells were infected with X4 tropic HIV-1 IIIB virus (20 ng/106 comparison with the NT siRNA-transfected cells (Fig. 2D, left cells). Two days later, HIV-1 p24 levels in the supernatant were measured panel). Furthermore, the SLP-76 knockdown cells also showed a by ELISA. Data are representative of three independent experiments. Virus reduced virus production with the R5 tropic HIV-1 virus (BaL production in the WT control was 4.2 6 0.3 ng/ml of p24 Ag. *p # 0.05 by guest on September 26, 2021 strain) (Fig. 2D, right panel), indicating that SLP-76 modulates versus WT cells. both X4 and R5 tropic virus infection in T cells. The above results underscore the role of SLP-76 in HIV-1 infection. SLP-76 does not regulate early steps of viral life cycle The NH2 acidic domain of SLP-76 regulates HIV-1 infection We further studied the mechanism by which SLP-76 regulates HIV- Three distinct domains of SLP-76—the amino terminal acidic 1 infection. For this, we first determined the steps in the HIV-1 life domain, the central proline-rich domain, and the C-terminal SH2 cycle that were regulated by SLP-76. The first step in the viral life domain—mediate protein interactions that are potentially impor- cycle is the binding of the viral envelope protein to the primary tant for its functions (6). A series of SLP-76 mutants schemati- receptor, CD4, and coreceptors, CXCR4 or CCR5. We have already cally represented in Fig. 3A has been generated previously (32) in demonstrated that SLP-76 expression does not regulate CD4 or which each mutant was designed to disrupt one of the three SLP- CXCR4 expression in Jurkat T cells (Fig. 1B). We tested whether 76 domains described. The first mutant, D1–156, consists of an in- the absence of SLP-76 expression specifically inhibited the frame deletion of the first 156 aa in the N-terminal region. The binding of HIV-1 virus as assessed by p24 Ag assay of the cell 2 2 second mutant, D224–244, contains a deletion of 20 aa in the lysates. There was no change in viral binding to SLP-76 / cells central region of SLP-76. The third mutant, R448K, contains compared with SLP-76+/+ and WT cells (Fig. 4A). Next, we a point mutation of a key arginine responsible for binding of specifically tested whether the absence of SLP-76 blocked cellular 2 2 phosphotyrosines to the SH2 domain of SLP-76 (Fig. 3A). To entry of HIV-1 in the WT, SLP-76+/+, and SLP-76 / cells using study the region of SLP-76 important for HIV-1 virus production, a HIV-1 virus entry assay. Again, we observed no change in the 2 2 we transfected these SLP-76 constructs (Δ1–156, Δ224–244, and amount of virus that enters the cell in SLP-76 / cells as com- R448K) into SLP-762/2 Jurkat T cells using the Amaxa Nu- pared with the WT and SLP-76+/+ control cells (Fig. 4B). Next, we cleofection Kit (Amaxa Biosystems). The transient expression of analyzed the role of SLP-76 in postentry events including first- different mutant constructs in SLP-762/2 cells was confirmed by strand cDNA synthesis and integration by PCR using primers as Western blot (Fig. 3B). The ability of HIV-1 IIIB to infect these shown in Table I. We did not observe any difference between HIV- 2 2 transfected cells in comparison with the WT and SLP-762/2 1–infected WT and SLP-76 / cells in both first-strand cDNA Jurkat cells was evaluated. As shown in Fig. 3C, transfection with synthesis (data not shown) and integration (Fig. 4C). Uninfected 2 2 Δ1–156 mutant showed ∼50% reduction in HIV-1 virus produc- WT and SLP76 / cells were used as controls (Fig. 4C). We tion, whereas the other mutants did not show any significant further verified the role of SLP-76 in HIV-1 entry and integration inhibition of HIV-1 virus production in comparison with WT- using an siRNA knockdown strategy in PBMCs. There was no infected cells. These studies suggest the importance of the NH2 change in viral entry and integration in SLP-76 siRNA-transfected terminal domain of SLP-76 in regulating HIV-1 infection. PBMCs compared with NT siRNA-transfected PBMCs (Fig. 4D, The Journal of Immunology 2773

FIGURE 4. SLP-76 does not inhibit viral binding, entry, integration, and transcription of HIV-1 in the T cells. (A) To analyze the effect of SLP-76 on virus binding, the various Jurkat T cell clones were infected with HIV-1 IIIB (40 ng/106 cells) and incubated for 1 h at 4˚C, and the cell lysates were analyzed for HIV-1 p24 levels. Virus level in the WT control was 263 6 20 pg/ml of p24 Ag. (B) To analyze the HIV-1 internali- zation, various Jurkat T cell clones were infected with HIV-1 IIIB (40 ng/106 cells) and incubated for 3 h at 37˚C, and cell lysates were then analyzed for the HIV-1 p24 levels by ELISA. Virus level in the WT control was 63 6 10 pg/ml of p24 Ag. (C) To analyze the effect of SLP-76 on integration, cells were infected with HIV-1 IIIB (20 ng/106 cells) for 24 h. DNA was extracted from the respective samples, and the integrated HIV-1 DNA 2/2 was analyzed by PCR. Uninfected WT and SLP-76 Downloaded from cells were used as controls. (D) To analyze the effect of SLP-76 on virus entry in PBMCs, the cells were transfected with NT and SLP-76 siRNAs. The trans- fected cells were infected with HIV-1 IIIB (40 ng/106 cells) at 37˚C for 3 h, and the cell lysates were analyzed for HIV-1 p24 levels. Virus level in the WT control was 230 6 20 pg/ml of p24 Ag. (E) NT and SLP-76 siRNA http://www.jimmunol.org/ knockdown PBMC samples were infected with HIV-1 IIIB (20 ng/106 cells) for 72 h, and DNA was used for the analysis of HIV integration. (F) To analyze the effect of SLP-76 on HIV transcription, WT and SLP-762/2 cells were infected with HIV and FL (left panel), and spliced RNA (right panel) transcription was evaluated by qRT-PCR. Data are representative of three indepen- dent experiments. by guest on September 26, 2021

4E). Altogether, these results indicate that upon infection with and SLP-762/2 cells, suggesting that SLP-76 does not regulate cell-free virus, viral binding, entry, and postentry events, including HIV transcription in T cells. integration, are not regulated by SLP-76. SLP-76 regulates release of HIV-1 from T cells SLP-76 does not regulate HIV-1 transcription We next analyzed whether SLP-76 modulates the release of virus We further evaluated the role of SLP-76 in regulating the post- using WT and SLP-762/2 Jurkat cells. WT and SLP-762/2 Jurkat integration steps prior to viral release. We quantitated the number T cells were infected with HIV-1 IIIB, and 72 h later, cells were of FL and spliced viral transcripts in WT and SLP-762/2 cells centrifuged, and the supernatant was collected and assayed for infected with HIV by qRT-PCR using primers as shown in Table I. virus release by HIV-1 p24 ELISA. Supernatant from SLP-762/2 There was no difference in FL (Fig. 4F, left panel) and spliced cells showed ∼60% reduction in extracellular p24 compared with mRNA levels (Fig. 4F, right panel) between HIV-1 infected WT the supernatants from WT cells (Fig. 5A). In addition, super- natants were also ultracentrifuged and the viral pellets immuno- blotted for p24. We observed significantly decreased expression 2/2 Table I. Primers used in the study (5-fold reduction) of HIV-1 p24 in the supernatants of SLP-76 cells in comparison with the supernatants of WT Jurkat cells (Fig. Target 5B). We also monitored the intracellular p24 levels in these cells Name Gene Sequence postinfection with HIV-1 IIIB by flow cytometry after 72 h of 9 ACCACAGTCCATGCCATCAC 9 infection. Interestingly, we did not observe any significant dif- GAPDH-F GAPDH 5 - -3 2 2 GAPDH-R 59-TCCACCACCCTGTTGCTGTA-39 ference in intracellular p24 levels in SLP-76 / cells compared MH535 Alu-LTR 59-AACTAGGGAACCCACTGCTTAAG-39 with WT cells (Fig. 5C). The above results suggest that virus 9 TGCTGGGATTACAGGCGTGAG 9 2 2 SB704 5 - -3 release is blocked in the SLP-76 / cells. HIV-FL F HIV-FL 59-CTGAAGCGCGCACGGCAA-39 gene SLP-76 is important for the cell–cell transmission of virus HIV-FL R 59-GACGCTCTCGCACCCATCTC-39 HIV-MS F HIV-MS 59-GACTCATCAAGTTTCTCTATCAAA-39 In addition to infection with cell-free virions, the importance of gene cell-associated spread across connecting membrane bridges and HIV-MS R 59-GTCTCTCAAGCGGTGGT-39 close cell–cell contacts referred to as virological synapse (VS) for F, forward; R, reverse. HIV-1 propagation is increasingly recognized and thought to 2774 SLP-76 REGULATES HIV-1 INFECTION

ratios (data not shown). Altogether, these results indicate that SLP-76 plays a key role in facilitating cell–cell transmission of virus. SLP-76 associates with Nef and modulates actin cytoskeletal reorganization We further studied the molecular mechanisms by which SLP-76 modulates HIV-1 infection. SLP-76, being an adaptor protein, associates with several molecules to mediate its functions. HIV-1 Nef is an important accessory protein in the virus that influences T cell activation and is known to interact with various molecules that mediate T cell activation (20, 21, 25, 26, 35). We therefore determined whether SLP-76 may mediate HIV-1 release by binding to Nef. The association of Nef with SLP-76 was first shown by overexpressing FLAG-tagged SLP-76 and Nef in 293 T cells (Fig. 6A). Next, we demonstrated the association of SLP- 76 and Nef in Nef-overexpressing Jurkat T cells by confocal microscopy (Fig. 6B, top panel). In addition, we also showed that

SLP-76 associates with Nef in HIV-1–infected Jurkat T cells (Fig. Downloaded from 6B, middle panel), but not in HIV-1–infected SLP-762/2 cells (Fig. 6B, bottom panel). Furthermore, we demonstrated the as- sociation between SLP-76 and Nef by immunoprecipitation in Nef-overexpressing Jurkat T cells (Fig. 6C) and HIV-1–infected Jurkat T cells (Fig. 6D). These results suggest that HIV-1 Nef may

associate directly with SLP-76. http://www.jimmunol.org/ Given that Nef and SLP-76 colocalize in T cells upon HIV-1 infection, it is reasonable to hypothesize that adapter protein SLP-76 may facilitate Nef’s function by modulating its association with other signaling molecules. A recent study has shown that a cooperative interaction of SLP-76 with Vav and Nck, both of which bind to the N-terminal domain of SLP-76 and are important for actin polymerization downstream of TCR (15). In addition, PAK2 has been shown to be a major Nef-associating protein that mediates its pathogenic effects. Therefore, we also determined the by guest on September 26, 2021 association of the SLP-76/Nef complex with PAK2, Vav1, and Nck1 in Nef-overexpressing Jurkat T cells. As shown in Fig. 7A, immunoprecipitation with Nef in Nef-overexpressing Jurkat cells revealed the association of Nef with PAK2, Vav1, and Nck1. In FIGURE 5. SLP-76 is essential for virus release and cell–cell trans- 2 2 addition, immunoprecipitation with SLP-76 in Nef-overexpressing mission. WT and SLP-76 / cells were infected with HIV-1 IIIB (20ng/ 106 cells), and supernatant was collected after 3 d of infection. (A) The Jurkat cells indicated that, in addition to Nef, SLP-76 also asso- HIV-1 p24 levels in the supernatant were determined by ELISA. Virus ciated with other signaling molecules including PAK2 and Nck1 production in the WT control was 3.3 6 0.1 ng/ml of p24 Ag. (B)In (Fig. 7B). We further evaluated the association of Nef with Nck1, 2 2 addition, the clarified supernatant was used for concentration of virus by Pak2, and Vav1 in HIV-1–infected SLP-76 / cells in comparison ultracentrifugation. The virus pellets were lysed and analyzed by Western with WT Jurkat cells. We observed that the association of Nef blotting (left panel) using p24-specific Ab. Amount of p24 expression in with Vav1, Pak2, and Nck1 was greatly reduced in SLP-762/2 the blot was estimated by densitometry (right panel). (C) The HIV-1– cells (Fig. 7C), suggesting that the association of Nef with these infected cells were fixed, permeabilized, stained for intracellular p24, and molecules is likely to be dependent on SLP-76. The above studies analyzed by flow cytometry. (D) Productively HIV-1–infected WT and 2 2 clearly suggest that the formation of a multimeric complex con- SLP-76 / cells (60–75% p24+ cells) were cocultivated with TZM-bl cells sisting of SLP-76, Nef, PAK2, Vav1, and Nck1 in HIV-1–infected at a 1:1 donor/target ratio. The donor cells were removed after 24 h, target cells were grown for another 72 h, and cell lysates were used to measure cells may regulate HIV-1 virus production. the luciferase activity. Data are representative of three independent HIV-1 transmission across the VS depends on cell polarization, experiments. *p # 0.05 versus WT cells. including reorganization of the actin cytoskeleton and recruitment of viral components to cell–cell contacts (33, 36). Because both constitute the predominant mechanism of HIV-1 propagation in SLP-76 and Nef have been shown to modulate actin polymeri- T lymphocyte cultures (33, 34). We further asked whether SLP-76 zation and cytoskeletal remodeling, we compared Nef-actin as- may affect direct cell-to-cell transmission of HIV-1. Briefly, WT sociation in WT and SLP-762/2 cells during HIV-1 infection by and SLP-762/2 Jurkat cells were first productively infected with confocal microscopy. As shown in Fig. 8, top panel, we observed HIV-1. After 3 d, ∼60–75% of the cells were HIV-1 p24 positive an association of Nef and F-actin in HIV-1–infected WT cells that in both SLP-762/2 and WT cells (data not shown); these cells was absent in HIV-1–infected SLP-762/2 cells. Pearson’s coef- were used as donors and cocultivated with TZM-bl cells (target ficiency values calculated using FV-10-ASW software (Olympus) cells) in various ratios: 1:1; 1:5, and 1:10. We observed that in indicated a strong Nef–F-actin association in WT cells that was comparison with WT cells, SLP-762/2 cells (used at ratio 1:1) absent in SLP-762/2 cells (Fig. 8, bottom panel). Based on our showed ∼30-fold reduction in cell–cell transfer to TZM-bl cells results, we hypothesize that SLP-76, through the formation of a (Fig. 5D). Similar kinetics were observed with other donor/target multimeric complex consisting of Nef and various host proteins The Journal of Immunology 2775

FIGURE 6. SLP-76 associates with HIV-1 Nef. (A) 293 T cells were transfected with FLAG-tagged SLP- 76, Nef, or control plasmids. (B) The cell lysates were immunoprecipitated with FLAG Ab and blotted with Nef (top panel) and SLP-76 Ab (bottom panel). Jurkat T cells expressing HIV-1 Nef were fixed, perme- abilized, and stained with anti-Nef and SLP-76 Abs followed by Alexa 488-tagged anti-mouse IgG Ab and Alexa-568 anti-rabbit IgG Ab (top panel). In addition, HIV-1 IIIB-infected WT (middle panel) or SLP-762/2 (bottom panel) were fixed, permeabilized, and stained with anti-Nef and SLP-76 Abs, followed by Alexa 488- tagged anti-mouse IgG Ab and Alexa-568 anti-rabbit IgG Ab. Slides were examined under Olympus FV1000 confocal microscope (633/1.4 oil; Olympus). The Downloaded from pictures were acquired using FV-10 ASW software. (C) Nef-expressing Jurkat T cells were lysed and immu- noprecipitated (IP) with anti-Nef Ab. The immune complexes were immunoblotted with SLP-76. (D) Uninfected (Un-Inf) and HIV-1–infected (HIV-Inf) WT Jurkat T cells were lysed and immunoprecipitated with Nef Ab and blotted with SLP-76 Ab. A vertical line http://www.jimmunol.org/ has been inserted to indicate a repositioned gel lane.

including Vav1, Nck1, and Pak2, may modulate actin cytoskeleton confirmed in MT4 cells and PBMCs using a SLP-76–specific

remodeling, which is ultimately important for the HIV-1 release. siRNA, suggesting that our findings are not cell line-specific or by guest on September 26, 2021 due to off-target effects. Discussion Our studies using mutants in various SLP-76 domains indicated Growing evidence suggests that TCR signaling complex plays that the N-terminal domain of SLP-76 was responsible for its a major role in regulating HIV-1 infection (1, 2). SLP-76 is a major modulation of HIV-1 infection in T cells. Structure-function studies adaptor molecule that links proximal and distal TCR signaling on SLP-76 have previously shown the importance of the N-terminal events through its function as a molecular scaffold in the assembly domain in TCR signaling (38, 39). The phosphotyrosines at aa 112 of multiprotein signaling complexes (37). In the current study, we and 128 in the N-terminal acidic region mediate binding to Vav1, demonstrate for the first time, to our knowledge, the prominent Nck1, and the p85 subunit of PI3K, whereas Tyr145 binds ITK (7– role played by SLP-76 in HIV-1 infection. Several lines of evi- 10). Other studies suggest that this region is a substrate of ZAP-70 dence presented in the study support this conclusion. In our (11). From our results, it appears that the binding of N-terminal studies using SLP-762/2, WT, and SLP-76+/+ (reconstituted) domain of SLP-76 to various other molecules may be important in Jurkat T cells, we found that the SLP-762/2 cell line was sig- positively regulating HIV-1 infection. In previous studies, ITK and nificantly resistant to HIV-1 infection. The role of SLP-76 was ZAP-70 have been shown to play a role in regulating HIV-1 in-

FIGURE 7. SLP-76/Nef form a signaling complex with PAK2, Vav1, and NCK1. (A) Nef-expressing Jurkat T cells (Nef+) were lysed and immuno- precipitated (IP) with anti-Nef Ab. The immune complexes were immunoblotted with PAK2, Vav1, NCK1, and Nef. (B) Nef-expressing Jurkat T cells (Nef+) were lysed and IP with anti–SLP-76 Ab. The immune complexes were immunoblotted with SLP-76, NCK1, PAK2, and Nef Abs. A vertical line has been inserted to indicate a repositioned gel lane. (C) HIV-1–infected WT and SLP-762/2 Jurkat T cells were lysed and IP with Nef Ab and blotted with NCK1, PAK2, Vav1, and Nef. Data are representative of three independent experiments. IgG represents control Ab, and TCL represents total cell lysates. 2776 SLP-76 REGULATES HIV-1 INFECTION

FIGURE 8. Nef–F-actin association is attenuated in HIV-1–infected SLP-762/2 cells. (A) HIV-1–infected WT and SLP-762/2 cells were fixed, permeabilized, and stained with anti-Nef Ab and Alexa Fluor 568– phalliodin (to analyze the F-actin polymerization) followed by Alexa 488-tagged anti-mouse IgG Ab. Slides were examined under Olympus FV1000 con- focal microscope (633/1.4 oil; Olympus). The pictures were acquired using FV-10 ASW software. The arrows shown point to actin-rich areas that associate with Nef. Data are representative of three independent experi- ments. (B) Nef-positive cells of both samples are se- lected by adjusting threshold of signal intensity, which Downloaded from are then counted as analyzing regions. Within ana- lyzing regions, Pearson’s coefficiency values of Nef and F-actin in WT and SLP-762/2 Jurkat cells are calculated with Olympus FV-1000 system (Olympus). p = 0.00017. Values are statistical summary of five fields. http://www.jimmunol.org/

fection in T cells (40, 41). ITK is a Tec family tyrosine kinase that Though the associations of Nef with several other molecules in by guest on September 26, 2021 binds to SLP-76 and functions as its downstream mediator in TCR the TCR signal transduction pathway have been shown (20, 21, signaling. A recent study demonstrating that ITK regulates viral 25, 26), this is the first study, to our knowledge, demonstrating entry, transcription, and assembly/release during HIV infection an association between SLP-76 and Nef. A large array of Nef- (40) may suggest that SLP-76 could be one of the upstream regulated effector functions are mediated by interactions of this mediators of ITK regulating virus replication. viral adaptor protein with several host cell factors in the TCR We also demonstrated that SLP-76 is required for cell–cell pathway (20, 21, 25, 26). We demonstrated that SLP-76 is involved transmission of HIV-1 using coculture of HIV-1–infected and in the formation of a multimeric complex consisting of Nef and noninfected cells. Cell–cell transmission of HIV-1, through the other host binding partners, including Vav1, Nck1, and Pak2. formation of a VS, is the predominant mode of HIV-1 transfer The manipulation of the actin cytoskeleton allows for efficient in vivo and probably occurs in secondary lymphoid organs, where cell–cell spread of the virus. An SLP-76 complex downstream of most lymphocytes are present and cells are in close contact with TCR has recently been shown to modulate actin cytoskeletal each other and with APCs (42). In addition to lymphocytes, reorganization in T cells (15). Furthermore, Nef has also been studies have shown that SLP-76 is expressed in other hemato- reported to mediate actin microfilament reorganization, which poietic cells, including monocytes/macrophages and dendritic in turn mediates viral release (17, 19, 20, 25–28). In the current cells that are important APCs for HIV-1 (5, 43). Our results with study, we showed reduced Nef–actin association in HIV-1–infec- T cells suggest that SLP-76 may also play an important role in ted SLP-762/2 cells in comparison with HIV-1–infected WT cells. transmission of HIV from these APCs to T cells. Though the role Furthermore, we have shown that SLP-76 associates with Nef and of SLP-76 has been studied in the formation of an immunological other signaling molecules. These data suggest that SLP-76 may synapse (44), our results suggest for the first time, to our knowl- form a multimeric complex consisting of Nef and other host cell edge, its role in the formation of a VS. factors that modulate actin cytoskeletal changes, which in turn SLP-76, being an adaptor protein, lacks intrinsic enzymatic ac- mediate viral release. tivity, serves as an essential scaffold for the construction of mul- Taken together, our studies, for the first time to our knowledge, timolecular protein complexes, and is indispensable for signal demonstrate a novel function of the adaptor molecule SLP-76 in transduction in several pathways including TCR activation (37). Our regulating HIV-1 virus production. These findings also provide next aim was to evaluate whether SLP-76 mediates HIV-1 infection new information on the modulation of the TCR signaling pathway by associating with viral proteins known to influence T cell acti- by HIV-1. Specifically, this study demonstrates the association of vation such as Nef. Using 293 T cells overexpressing Nef and SLP- SLP-76 with the viral protein Nef and its role in modulating actin 76, Nef-overexpressing Jurkat T cells, and HIV-1–infected cells, cytoskeletal remodeling. Our findings suggest that the formation we could demonstrate an association between SLP-76 and Nef. of the immunological synapse and VS may involve common The Journal of Immunology 2777 signaling molecules such as SLP-76. Therefore, targeting SLP-76 20. Fackler, O. T., W. Luo, M. Geyer, A. S. Alberts, and B. M. Peterlin. 1999. Activation of Vav by Nef induces cytoskeletal rearrangements and downstream or its interaction with Nef may represent an attractive target for effector functions. Mol. Cell 3: 729–739. the future therapeutic interventions against HIV-1. 21. Manninen, A., and K. Saksela. 2002. HIV-1 Nef interacts with inositol tri- sphosphate receptor to activate in T cells. J. Exp. Med. 195: 1023–1032. 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