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c-Src and Pyk2 Protein Tyrosine Kinases Play Protective Roles in Early HIV-1 Infection of CD4+ T-Cell Lines

McCarthy, Stephen D.S. BSc*; Jung, Daniel PhD; Sakac, Darinka MS‡,§; Branch, Donald R. PhD*,‡,§

JAIDS Journal of Acquired Immune Deficiency Syndromes: 1 June 2014 - Volume 66 - Issue 2 - p 118–126
doi: 10.1097/QAI.0000000000000105
Basic and Translational Science

Background: During early HIV-1 infection of CD4+ T-lymphocytes, many host protein tyrosine kinases become activated within minutes, including phosphoprotein pp60c-src (c-Src) and the focal adhesion kinase family member, proline-rich tyrosine kinase 2 (Pyk2). Whether their activation facilitates or impedes infection remains to be determined.

Methods: c-Src kinase inhibitors (SU6656, PP1, and PP2), adenovectors [wild-type and dominant-negative (DN) c-src] or siRNA (targeting c-src or pyk2) were used to inhibit, compete with or knockdown c-Src in Jurkat C, Jurkat E6-1, Hut 78 or Kit225 T-cell lines. Cells were then infected with HIV-1 luciferase reporter virus expressing VSV-G or HXB2(X4) envelope, and luciferase activity was measured after 2 days. Reverse transcriptase activity and viral cDNA were measured 1 hour after infection, whereas integrated virus was measured 12 hours after infection.

Results: Pretreating Jurkat T-cells with SU6656 led to increased VSV-G luciferase activity. In the adenovector experiments, T-cells overexpressing dominant-negative c-Src, but not wild-type c-Src, showed increased luciferase activity after VSV-G infection. siRNA knockdown of c-Src or Pyk2, followed by HXB2 infection in Jurkat T-cells, lead to increased reverse transcriptase activity, viral cDNA, integrated virus, and increased luciferase activity.

Conclusions: Pyk2 is known to interact with c-Src. Thus, Pyk2 activation that coincides with increased c-Src activity during HIV-1 infection could be responsible for c-Src activation. Reduced c-Src activation increases HIV-1 reverse transcription, integration, and/or transcription, suggesting the high c-Src activity seen early in HIV-1 infection may be a cellular response to slow or prevent early infection in CD4+ T-cells.

*Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada;

Department of Microbiology and Biochemistry, Laval University, Québec, Canada;

Centre for Innovation, Canadian Blood Services, Toronto, Canada; and

§Division of Advanced Diagnostics, Infection and Immunity Group, Toronto General Research Institute, Toronto, Canada.

Correspondence to: Donald R. Branch, PhD, Centre for Innovation, Canadian Blood Services, Room 342, 67 College Street, Toronto, Ontario, Canada, M5G 2M1 (e-mail:

Presented at the XIX International AIDS Conference, July 22–27, 2012, Washington, DC, and 21st Annual Canadian Conference on HIV/AIDS Research, April 19–22, 2012, Montreal, Quebec, Canada.

Supported by Ontario HIV Treatment Network (OHTN) and the Canadian Foundation for AIDS Research (CANFAR).

D.R.B. is a founding shareholder in ViroCarb Inc. Vanier CGS D and NSERC CGS M scholarships awarded to S.D.S.M. The remaining authors have no conflicts of interest to disclose.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (

Received December 31, 2013

Accepted December 31, 2013

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Chronic immune activation and T-cell dysregulation persist during asymptomatic HIV-1 infection, and yet we still do not have a clear understanding of the early stages of these events.1 From the initial binding of viral glycoprotein 120 (gp120) to T-lymphocyte receptor CD4 and chemokine coreceptors CXCR4 or CCR5, signaling cascades are induced that promote viral infection.2 It is known that tyrosine kinases are major regulators of HIV-1 infection because the pan-tyrosine kinase inhibitor, genistein, inhibits HIV-1 infection in macrophages.3 Moreover, HIV-1–infected patients show defective early protein tyrosine phosphorylation in peripheral blood mononuclear cells4 and in CD4+ T-cells specifically.5 Indeed, much research has been done on the proximal signaling cascades induced on viral binding and fusion, which interfere with normal T-cell activation and cortical actin rearrangement.6 However, a functional role for many kinases hijacked immediately after viral entry remains to be discovered.

Nonreceptor tyrosine kinases play critical roles throughout the HIV-1 lifecycle in T-cells. Changes in tyrosine phosphorylation signaling are necessary for viral entry,7 actin remodeling,6,8 viral RNA reverse transcription,8 translocation of the viral preintegration complex (PIC) to the nucleus, viral integration,9 viral DNA transcription, and viral egress.10 In particular, HIV-1–infected T-cells show striking changes in the activity of the Src-family of tyrosine kinases.11 They become activated within minutes of HIV-1 infection; however, their roles at this early time point are only partially understood. There are 9 Src-family members, 4 of which are expressed in T-cells: Lck, c-Src, Fyn, and c-Yes. Lck has been the most studied of these 4 in terms of early HIV-1 infection,5,7,12 as it is a proximal signaling molecule directly associated with CD4 and is critical for T-cell activation and growth.13 Increased Lck activity was found to reduce viral replication in various T-cell lines.7 In addition, increased Fyn activity correlates with greater recruitment of tyrosine-phosphorylated APOBEC3G into HIV-1 particles,14 corroborating our previous observation of higher Fyn activity in patients with asymptomatic HIV-1 infection.4 Clearly, the Src-family of protein kinases is implicated in HIV-1 infection, but the functional role of activated c-Src during early HIV-1 entry remains unknown.

Previously, we have shown that negligible c-Src protein exists in resting peripheral blood T-lymphocytes, but that it is produced within 12 hours of T-cell stimulation.15 c-Src phosphorylates more than 64 known cellular substrates, activating signaling cascades involved in cell migration, cytoskeletal rearrangement, cell proliferation, and cell survival.16 The enzyme often associates with cell membranes, in particular at focal adhesions of the plasma membrane and the perinuclear golgi region.17 When HIV-1 initially attaches to the host membrane, viral gp120 is able to bind the G protein-coupled receptor CXCR4 or CCR5, inducing the autophosphorylation of proline-rich tyrosine kinase 2 (Pyk2 or FAK2) within the cell.2 Pyk2 plays an important role at focal adhesions at the cell periphery and in cytoskeleton remodeling. Activated Pyk2 can then recruit the binding of the c-Src src-homology-2 (SH2) domain, which allows c-Src to be autophosphorylated at Tyr419, activating the enzyme.18,19 Both of these kinases become phosphorylated within minutes of HIV-1 infection. A recent study has shown that Pyk2 shRNA knockdown in CEM T-cells reduced viral p24 production within 6 days of HIV-1NDK infection.20 Moreover, Gilbert et al. showed that inhibiting c-Src with the kinase inhibitor PP2 caused a dramatic decrease in p24 production after 6 days of infection.21 However, the roles of Pyk2 and c-Src in the first few hours of early HIV-1 infection, when these enzymes are first activated, are not well defined.

We hypothesized that c-Src and Pyk2 nonreceptor tyrosine kinases play an important role in facilitating early HIV-1 infection of CD4+ T-cells. By using c-Src drug inhibitors, a dominant-negative (DN) c-Src construct and siRNA knockdown, we were surprised to find that reducing c-Src protein levels or its activity caused an increase in viral infection in 3 laboratory T-cell lines.

This research is significant because small molecular kinase inhibitors targeting c-Src may not be efficacious in reducing initial HIV-1 infection, but may be valuable in studying the formation and/or stability of the HIV-1 reverse transcriptase complex and PIC.

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Cells and Cell Culture Conditions

In these in vitro experiments, we used the human T-cell leukemia cell lines Jurkat C (from Jurkat-FHCRC, a gift from Dr. G. B. Mills, MD Anderson Cancer Center, Houston, TX), Jurkat E6-1, and a cutaneous T-lymphocyte cell line Hut 78 from the American Type Culture Collection (ATCC, Rockville, MD). We also used a chronic lymphocytic leukemia T-cell line, Kit 225, also a gift from Dr. G. B. Mills. These T-cell lines were grown in complete medium consisting of RPMI-1640 with L-glutamine and NaHCO3 (SIGMA, St. Louis, MO), supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS; Wisent, Saint-Jean-Baptiste, Canada), 100 units per milliliter penicillin (Gibco, Burlington, Canada), 100 μg/mL streptomycin (Gibco), 10 μg/mL gentamycin (Gibco), and 10 units/mL human recombinant IL-2 (Kit 225 cells only; SIGMA). Human embryonic kidney cells (HEK 293T, ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with the same proportion of FBS and antibiotics as above. MT-4 cells from the NIH AIDS Research and Reference Reagent Program (Germantown, Philadelphia, PA) were used to determine viral multiplicity of infection (MOI). All cells were incubated at 37°C in a 5% CO2 atmosphere.

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Generation of Infectious HIV-1, VSV-G Pseudoenveloped, and HXB2 Enveloped Virus

In brief, X4 HIV-1IIIB from the NIH AIDS Research and Reference Reagent Program (Rockville) was grown in Jurkat C cells (1 × 106 cells/mL) in a biosafety level 3 (BSL3) laboratory.22 The cell supernatant was collected, viral stocks were titrated and assessed by p24 antigen enzyme-linked immunosorbent assay (ZeptoMetrix, Buffalo, NY), and aliquots were frozen at −80°C. MOI was determined using MT-4 cells.22

We produced recombinant, replication deficient VSV-G pseudoenveloped or HXB2 enveloped (X4) HIV-1 containing a luciferase gene, as described previously.23 Briefly, 2.5 × 106 HEK 293T cells were plated with 10 mL of DMEM 24 hours before transfection. We cotransfected 15 μg of HIV-1 NL4-3luc (luciferase gene inserted into viral nef gene and env deficient, a kind gift from Dr. Veneet KewalRamani, New York Medical University, NY) with 10 μg of VSV-G env plasmid (amphotropic envelope of the vesicular stomatitis virus, a generous gift from Dr. M. Tremblay, Quebec City, PQ) into each 10-cm plate of HEK 293T cells with the CalPhos Mammalian Transfection Kit (Clontech, Mountain View, CA), following the manufacturer's instructions. The same was performed for the HXB2 plasmid (HIV-1 NL4-3luc lacking nef and containing HXB2 (X4) env, another generous gift from Dr. M. Tremblay) at 15 μg per plate. Dulbecco's modified eagle medium was replaced after 25 hours, and viral supernatant was collected on days 2 and 3. The supernatant was syringed through 0.45-μm pore filters (Millipore, Billerica, MA), concentrated by underlaying with a 20% sucrose gradient, and centrifuged at 16,000 × g for 90 minutes at 4°C. Virus pellet was resuspended in TNE buffer (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, and 100 mM NaCl) and stored in −80°C aliquots. p24 levels were measured by enzyme-linked immunosorbent assay. All viruses underwent 1 freeze–thaw cycle before infection studies.

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c-Src Drug Inhibition

Before infection, 2 × 106 T-cells were inhibited with 5–20 uM of SU6656 (In Solution), 10 uM PP1 (Analog) or 10 uM PP2 (Calbiochem, La Jolla, CA) for 1 hour, washed with Dulbecco's Phosphate-Buffered Saline (PBS) lacking calcium chloride and magnesium chloride (SIGMA), resuspended in RPMI media, and then counted again. The solvent Dimethyl Sulfoxide (DMSO, Bioshop, Burlington, Canada) was used as a negative control for SU6656.

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Kinase Assay

Jurkat C cells treated for 1 hour with SU6656 were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM HEPES, pH 7.3, 1% Nonidet P-40, 0.1% SDS, 0.1% Na3 deoxycholate, 150 mM NaCl, 1 mM Na3VO4, 50 M ZnCl2, 2 mM EDTA, and 2 mM PMSF) and immunoprecipitated with anti-c-Src clone 327 (Oncogene, Cambridge, MA). Lysates and beads were washed and resuspended in kinase buffer, including 5 uCi γ32P-ATP (MP Biomedicals, Santa Ana, CA) and heat-inactivated rabbit enolase (MP Biomedicals) as substrate. Protein samples were boiled for 10 minutes and then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% reducing gel) and transferred to an Immobilon-P membrane (Millipore). Enolase phosphorylation by c-Src was determined by autoradiography. In vitro kinase activity was measured as the densitometry of enolase activity divided by the amount of c-Src per lane and normalized to the DMSO control treatment.

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c-Src Adenovirus Gene Transduction

Recombinant Ad5/F35 adenovirus vectors were created by Daniel Jung. These bicistronic vectors contained enhanced yellow fluorescent protein (EYFP) and expressed wild-type (WT) human c-Src, a dominant negative (DN), kinase inactive c-Src or no c-Src as a control (empty vector; EV).24 Cells were titrated with virus to determine the highest MOI that permitted 70%–80% gene transduction, while minimizing cell death 48 hours after infection. EYFP detection and viability staining (7-AAD, which stains both apoptotic cells and necrotic bodies, BD Sciences, Franklin Lakes, NJ) were performed on fixed cells using FACS (see Flow Cytometry and Data Acquisition).

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siRNA Knockdown

Pools of 4 siRNA duplexes, specific for human c-Src (cat # M-003175-03) or Pyk2 (cat # M-003164-02) mRNA, and a control pool of nontargeting siRNA (cat # D-001206-13-05) were ordered from Dharmacon RNAi technologies (siGENOME SMART Pool; Thermo Scientific, Lafayette, LA). In Jurkat E6-1 cells, siRNA knockdown was achieved using the siRNA cationic lipid transfection reagent GeneSilencer (Genlantis, San Diego, CA), following the manufacturer's instructions. Two microliter of siGuard RNase inhibitor (Genlantis) was added to each 1-mL reaction to prevent RNAse degradation of the RNA duplexes. On siRNA titration, 900 nM was determined to give the optimal level of protein knockdown for both c-Src and Pyk2 through Western blot densitometry at 48 hours.

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HIV-1 Infection and Luciferase Assay

1 × 106 T-cells pretreated with drug, adenovector or siRNA were infected with 1.4 ng of VSV-G/HIV-1 or 7.5 ng of HXB2 at 37°C in 2 mL of growth media in 12-well plates for 2 days. Cells were counted, whereas others were lysed with the Luciferase Assay System (Promega, Madison, WI) or kept for Western blots.

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Jurkat C cells were serum starved and then, immediately after 15 minutes of HIV-1IIIB infection, were washed in PBS and lysed with RIPA buffer. Monoclonal antiphosphotyrosine (anti-ptyr) clone 4G10 (Millipore) and protein G-Sepharose beads (Santa Cruz Biotechnology, Dallas, TX) were used to immunopreciptate phosphoproteins from 4 × 106 Jurkat C cell lysates.

After VSV-G/HIV-1 or HXB2 infection, 5–10 × 105 T-cells were washed in PBS and lysed with RIPA buffer.

After boiling samples, proteins were resolved on an 8%, reducing SDS-PAGE gel and transferred to an Immobilon-P membrane for Western blot detection. The blots were blocked with 5% skimmed-milk powder containing 0.5% Nonidet P-40 (BioShop, Burlington, Canada). Primary antibodies diluted in 5% milk and 0.5% Nonidet P-40, anti-β-actin (Sigma), anti-α-tubulin (Sigma) anti-Pyk2 (Transduction Laboratories), and anti-c-Src GD11 (Millipore) were used to probe the blots. After washing, secondary goat anti-mouse HRP antibody (Bio-Rad, Mississauga, Canada) was added, followed by enhanced chemilluminescent (ECL) detection (GE Healthcare, Buckinghamshire, United Kingdom). Pyk2 and c-Src protein levels were determined by comparing their densitometry to normalizers β-actin or α-tubulin with the Molecular Imager GelDoc XR+ Imagin System (Bio-Rad).

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DNA Isolation and Quantitative Polymerase Chain Reaction

Genomic and viral cDNA was isolated from 5 × 105 Jurkat E6-1 cells, pretreated by siRNA transfection, and infected with HXB2 virus for 1 or 12 hours, using the QIAamp DNA Blood Mini Kit (Qiagen, Toronto, Canada). Samples were heated at 56°C for 2 hours in QIAamp DNA Blood Mini Kit buffer AL to deactivate viable viral particles.

The primer sets used to detect human or viral DNA (synthesized by the Center for Applied Genomics, The Hospital for Sick Children, Toronto, Canada) were as follows25–27: β-globin forward, 5′-CCCTTGGACCCAGAGGTTCT-3′; β-globin reverse, 5′-CGAGCACTTTCTTGCCATGA-3′; early reverse transcripts (early RT) forward, 5′-GTAACTAGAGATCCCTCAGACCCTTTTAG-3′; early RT reverse, 5′-TAGCAGTGGCGCCCGA-3′; late reverse transcripts (late RT) forward, 5′-CCGTCTGTTGTGTGACTCTGG-3′; late RT reverse, 5′-GAGTCCTGCGTCGAGAGATCT-3′; genomic Alu forward, 5′-GCCTCCCAAAGTGCTGGGATTACAG-3′; HIV-1 gag reverse, 5′-GCTCTCGCACCCATCTCTCTCC-3′; HIV-1 LTR forward, 5′-GCCTCAATAAAGCTTGCCTTGA-3′; HIV-1 LTR reverse, 5′-TCCACACTGACTAAAAGGGTCTGA-3′. For each quantitative polymerase chain reaction (qPCR) run, 50–100 ng template DNA (with the exception of the no-template control, NTC) was added to polymerase chain reaction (PCR) tubes containing 12.5 μL 2× SYBR Green PCR Master Mix (Applied Biosystems), 300–900 nM of each forward and reverse primers, and PCR grade H2O (Roche Diagnostics, Indianapolis, IN) up to a final volume of 25 μL. PCR cycling parameters were as follows: initial denaturation at 95°C for 10 minutes; 40–50 cycles of amplification of 95°C for 15 seconds, 58°C for 30 seconds (β-globin or late RT, 54°C for early RT) and 60°C for 30 seconds. To measure integrated virus by Alu-gag PCR,27 tubes were first preamplified with 100 nM Alu forward and 600 nM gag reverse primers, with 2× SYBR Green PCR Master Mix as above, for 10 cycles of 93°C for 30 seconds, 50°C for 60 seconds, and 60°C for 100 seconds. Nested PCR was performed on these samples by adding 300 nM each of HIV-1 LTR forward and reverse primers, with 2× SYBR Green PCR Master Mix, for 40 more cycles using the above parameters. Duplicate reactions were analyzed using the Rotor-Gene RG-3000 thermocycler (Corbett Research, Montreal, Canada). c-Src and Pyk2 siRNA-treated groups were compared with the nontargeting siRNA-treated group by the 2-ΔΔCT comparative CT method.28

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Flow Cytometry and Data Acquisition

Cell surface expression of CD4 and CXCR4 were determined on 2.5 × 105 Jurkat E6-1 cells per FACS tube, 48 hours after siRNA knockdown. Cell surface staining was performed as previously described.29 In brief, cells were stained with 5 μL of FITC mouse antihuman CD4, 5 μL of PE mouse antihuman CXCR4, and 2.5 μL of 7-AAD in 100 μL of FACS buffer (PBS + 2.5% FBS). Isotype control antibodies were FITC-conjugated mouse IgG1κ and PE-conjugated mouse IgG2aκ. All of these antibodies were purchased from BD Pharmingen, Mississauga, Canada. Data on 20,000 events per tube were collected using Becton Dickenson FACSCalibur (BD Calibrite; BD Biosciences). Signals were acquired for the forward scatter, side scatter, the green (FITC, EYFP), yellow (PE), and red (7-AAD) channels.

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Reverse Transcriptase Activity Assay

Quantification of reverse transcriptase activity from infected cell lysates was performed using the SYBR Green I qPCR-based product-enhanced RT (PERT) assay.30 Briefly, 5 × 105 JE6-1 cells were infected with HXB2 virus for 1 hour, washed twice with PBS, lysed, diluted 10-fold with 10× sample dilution buffer, and given 1 μg of MS2 RNA template (Roche Diagnostics) to reverse transcribe in vitro. The cDNA product was measured by qPCR, and a standard curve of recombinant HIV-1 RT (Calbiochem) was used to assign enzyme activity units to reverse transcriptase from the infected cell lysate samples.

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Statistical Analysis

Mean values were compared using a two-tailed, unpaired Student's t-test and corrected for multiple comparisons when more than two mean values were considered in an experiment, to minimize the probability of type 1 errors. FACS data show the median fluorescent intensity and percent-positive cells collected with CellQuest software (BD Biosciences) and analyzed by FlowJo version X software. Comparisons of histogram EYFP expression were performed with the 2 samples Kolmogorov–Smirnov test. For all figures, an asterisk (*) denotes a P value < 0.05, 2 asterisks (**) denotes a P value < 0.01, and 3 asterisks (***) denotes a P value < 0.005. Error bars shown are the standard error around the mean.

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Tyrosine Kinases Become Activated Within Minutes of HIV-1 Infection

To confirm previous reports that nonreceptor kinases become activated within minutes of HIV-1 infection, we infected serum-starved Jurkat C T-cells with X4 strains HIV-1IIIB and HXB2 or with VSV-G pseudoenveloped HIV-1 virus. We then immunoprecipitated protein substrates from T-cell lysates with anti-ptyr clone 4G10 (Fig. 1). Probing the blot of HIV-1IIIB–infected cell lysate with anti-ptyr revealed a robust phosphorylation of substrates in the 55–130 kDa range and 1 low molecular band at ∼40–42 kDa (Fig. 1A). Reprobing the Western blot for Pyk2 and c-Src (Fig. 1B), demonstrated phosphorylation of these kinases within 15 minutes of HIV-1 infection, indicative of their activation and recruitment during early HIV-1 entry events.

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SU6656 Consistently Inhibited c-Src Activity and Increased VSV-G/HIV-1 Infection

To directly implicate a role for c-Src activity during early HIV-1 infection, we used well-known c-Src inhibitors (PP1, PP2, and SU6656)31 to inhibit c-Src activity before HIV-1 infection. For these experiments, a replication deficient, pseudotyped nef-deficient VSV-G/HIV-1 virus carrying a luciferase reporter gene was used, so as not to produce viral protein products, which are known to directly bind and activate the Src-family of kinases, such as viral protein nef.32 After pretreating Jurkat E6-1 cells with 10 uM of drug for 1 hour, only the SU6656-treated cells showed a significant increase in VSV-G/HIV-1 infection after 2 days, compared with DMSO control (Fig. 2A). Cell growth was not adversely affected during this experiment (see Figure S1A, Supplemental Digital Content, The nonspecific effects of SU6656 differ from PP1 and PP231, which is why we decided to move forward using SU6656 as our c-Src inhibitor. Our in vitro kinase assay showed that SU6656 reduces c-Src activity by 50% at 10 uM (Fig. 2B). We repeated our VSV-G/HIV-1 infection experiment at 2 different SU6656 drug concentrations (Fig. 2C) and in another Jurkat T-cell line (Fig. 2D) to confirm our finding that c-Src drug inhibition leads to increased VSV-G/HIV-1 infection in T-cell lines.

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Adenovector Expressing DN c-Src Increased VSV-G/HIV-1 Infection in CD4+ T-Cell Lines

As with most kinase inhibitors, we cannot ignore that SU6656 inhibits related Src-family members, such as Lck and Fyn, in conjunction with c-Src. Thus, we used adenovirus gene transduction to alter the pool of functional c-Src in 3 T-cell lines before VSV-G/HIV-1 infection. Jurkat E6-1, Hut 78, and Kit 225 T-cells were transduced with adenoviruses containing an EV control, WT c-Src or a DN c-Src that can bind but not phosphorylate substrate (Fig. 3). Administering adenovector expressing WT c-Src or DN c-Src did not alter cell survival when compared with EV, within each cell type (see Figure S1B, Supplemental Digital Content, Once we optimized MOI to reduce cell death and maintain high gene transduction, we achieved 62%–84% EYFP expression in Jurkat E6-1 and Hut 78 cells after 2 days (Figs. 3B–C). Kit 225 cells were less transducible (36%–47%, Fig. 3D) but showed higher overall survival. No statistical difference was found between EV, WT c-Src, or DN c-Src EYFP expression by the Kolmogorov–Smirnov test.

Viral infection was normalized to the total cells in each well after 2 days of VSV-G/HIV-1 infection. At this time point, no significant difference in VSV-G/HIV-1 infectivity was found between EV and WT c-Src treatments (Figs. 3E–G). However, administering the DN c-Src adenovector caused a significant increase in VSV-G/HIV-1 infection in 2 of the 3 T-cell lines tested. This finding complements our previous c-Src drug experiments. Competing endogenous c-Src enzyme with the DN mutant increases the integration or transcription of VSV-G/HIV-1.

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Both c-Src and Pyk2 Knockdown Caused an Increase in Either VSV-G/HIV-1 or HXB2 Infection

To assess whether the upstream binding partner Pyk2 mediates c-Src's effects during HIV-1 infection, we used siRNA to knockdown mRNA expression of either kinase in Jurkat E6-1 cells. Relative to a nontargeting siRNA control, cell proliferation was not affected by Pyk2 or c-Src knockdown (see Figure S2A, Supplemental Digital Content, T-cells are a challenging nonadherent cell line to transfect for siRNA knockdown, hence we optimized the siRNA concentration delivered and the time point to assess protein knockdown by Western blot (see Figure S2, Supplemental Digital Content, Qualitatively, we found 900 nM of siRNA transfected by lipofection could reliably knockdown either c-Src or Pyk2 by 40%–50%, 48 hours after transfection (Figs. 4A–D).

At this time point, we then infected the Jurkat E6-1 cells with VSV-G/HIV-1 and measured infection 2 days later. Interestingly, both c-Src and Pyk2 knockdown in Jurkat E6-1 cells lead to an increase in luciferase activity on infection with the VSV-G/HIV-1 reporter virus, when compared with the nontargeting siRNA control group (Fig. 4E). This implicates both kinases as having important catalytic activity during early HIV-1 infection of T-cells. To determine whether this phenomenon can be attributed to the differences in vesicular stomatitis entry into a host cell (clatherin-dependant endocytosis)33 from that of HIV-1 (receptor-mediated internalization), we performed the siRNA knockdown experiment once more with an HXB2 (X4) luciferase reporter virus that is also replication deficient. Similar to the VSV-G/HIV-1 infection, when either c-Src or Pyk2 protein expression is reduced by siRNA knockdown, we saw a concomitant increase in HXB2 viral infection after 2 days (Fig. 4F). This suggests that Pyk2 and c-Src may play similar roles in the early HIV-1 entry events of both viruses downstream of HIV-1 receptor binding and fusion.

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c-Src and Pyk2 siRNA Knockdown Increased the Reverse Transcription of HXB2 cDNA

To assess at which early stage c-Src or Pyk2 were having their effect, we measured the production of HIV-1 early RT, late RT, and integrated virus, shortly after HXB2 infection by real-time qPCR (Fig. 5A). One hour after infection, both c-Src and Pyk2 siRNA knockdown led to a 4–10 fold increase in early and late reverse transcripts, relative to nontargeting siRNA-treated cells. Knocking down c-Src or Pyk2 also led to a 3–4 fold increase in integrated virus measured after 12 hours of infection. We detected no increase in CD4 or CXCR4 cell surface expression after c-Src or Pyk2 knockdown (Fig. 5B), suggesting the increase in HIV-1 early RT, late RT and integrated virus is not due to increased receptor binding on entry. Measuring reverse transcriptase activity 1 hour after infection revealed increased activity from lysates of Jurkat cells administered c-Src or Pyk2 siRNA (Fig. 5C), suggesting that they act, at least in part, at the level of HIV-1 reverse transcription.

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This study demonstrates that reducing c-Src enzyme activity, outcompeting endogenous c-Src with a DN Src mutant, or reducing c-Src protein levels with siRNA knockdown caused an increase in VSV-G/HIV-1 infection in various T-cell lines. We first demonstrated that c-Src becomes activated within 15 minutes of HIV-1 infection, which agrees with our previous findings.11 We also found that Pyk2 becomes phosphorylated in the same 15 minutes time span, faster than Seror et al.20 reported using HIV-1NDK virus.

Reducing c-Src activity with SU6656 consistently caused an increase in VSV-G/HIV-1 infection in Jurkat T-cells. Initially, this finding seems paradoxical considering that CD4+ T-lymphocytes pretreated with PP2 were previously reported to show a decrease in p24 levels 6 days after NL4-3balenv or JR-CSF (R5) infection.21 This discrepancy may be because our viral read-out measured transcription of luciferase to detect successful viral integration, instead of p24 to monitor viral production and egress. The initial activation of nonreceptor tyrosine kinases is rapid,11 and kinases that play a role during early entry may have different roles during viral egress and p24 release. Thus, inhibiting c-Src activity may promote early entry of HIV-1, as we have found, and yet reduce viral p24 production at later stages of the viral replication cycle. This difference in function could perhaps be attributed to the downstream substrate, the 68 kDa Src-associated protein in mitosis (Sam 68), which is known to assist in the nuclear export and translation of HIV-1 RNA.34

We also observed that the addition of adenovector producing DN c-Src in 3 T-cell lines caused an increase in VSV-G/HIV-1 infection, yet adding adenovector expressing wild type c-Src did not reduce infection. Perhaps this can be attributed to the dynamic regulation of c-Src by cellular phosphatases (SHP-1, CD45) and kinases (Csk), explaining why the addition of wild type c-Src could not reduce viral infection. Nonetheless, the ability of DN c-Src to compete with endogenous c-Src for substrates and cause an increase in VSV-G/HIV-1 infection agrees well with our drug experiments. Whether c-Src directly phosphorylates HIV-1 proteins or triggers signaling pathways that hinder viral entry and integration warrants further investigation.

In terms of our siRNA knockdown findings, both c-Src and Pyk2 knockdown caused an increase in VSV-G/HIV-1 infection in Jurkat cells. This agrees well with the previous finding that activated Pyk2 links G protein-coupled receptors to downstream signaling by complexing with c-Src and subsequently causing c-Src activation.19 It is interesting to note that while VSV-G/HIV-1 enters cells through clatherin-dependent endocytosis,13 using a virus with HXB2 (X4) envelope gave a similar result, suggesting both kinases play important roles in HIV-1 infection regardless of initial entry route. We also found increased reverse transcriptase activity, early reverse transcripts, late reverse transcripts, and integrated virus after c-Src or Pyk2 siRNA knockdown. Pyk2, being upstream of c-Src, had a stronger effect on viral DNA produced in cells. It will be of interest to determine the mechanism by which these kinases affect early infection, perhaps stabilizing the reverse transcriptase complex or PIC through phosphorylation or allosteric interactions.

Our research is the first study to explore the impact of c-Src and Pyk2 activation on early HIV-1 infection in CD4+ T-cell lines. Discovering novel mechanisms in the entry of HIV-1 and their impact on T-cell signal transduction may lead to targeting these pathways with drugs and improving the treatment options available to people with HIV-1. Tyrosine kinase inhibitors with low-toxicity profiles exist for cancer therapies and could be repositioned as viable options for treating HIV-1 infection. For instance, cyclin-dependant kinase inhibitors such as r-roscovitine and alsterpaullone are showing promising anti-HIV-1 properties in peripheral blood mononuclear cells by preventing viral Tat transactivation.35 We also know that tyrosine kinase inhibitors can be safe in HIV-1 patients, as a case study of patients with AIDS-related Kaposi Sarcoma given the Abl kinase inhibitor imatinib showed no increase in viral plasma load.36 Our results show increased HIV-1 early reverse transcripts, late reverse transcripts, and integration when c-Src or Pyk2 are inhibited, which may preclude the use of Src inhibitors during early HIV-1 infection. However, T-cell lines are transformed cells and do not necessarily represent what would be the findings using primary CD4+ T-cells isolated from normal donors. These studies are currently in progress.

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The authors thank Yulia Katsman, Amanda Harrison Wong, Evgenia Bloch, Iain Scovell, Marie Pier Cayer, and Danila Leontyev for their assistance with experiments.

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nonreceptor tyrosine kinases; pp60c-src; Pyk2; CD4+ T-cells; HIV-1 entry; siRNA knockdown

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