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Introduction

The Pyk2 and c-Src protein tyrosine kinases play protective roles in early HIV-1 infection of CD4 + T-cells .

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Introduction

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  1. The Pyk2 and c-Src protein tyrosine kinases play protective roles in early HIV-1 infection of CD4+ T-cells Stephen D.S. McCarthy, Darinka Sakac, Xue-Zhong Ma, Daniel Jung and Donald R. Branch. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Introduction Methods (cont’d) Results (cont’d) The human immunodeficiency virus type 1 (HIV-1) up-regulates many host kinases upon infecting CD4+ T-cells. Aberrant signaling may explain aspects of CD4+ T-cell dysregulation and chronic immune activation. Although phosphoprotein c-Src (pp60c-Src) and proline rich tyrosine kinase 2 (Pyk2) become activated during HIV-1 infection, the role of their signaling cascades shortly after viral entry are not well defined. Receptor binding induces kinase signaling cascades 4. Knocking down c-Src or Pyk2 leads to increased HXB2 infection in JE6-1. A) Luciferase Activity B) Protein Densitometry *** *** 1.0 5000 RLU (units) X-fold change Results 0.5 0 0 1. HIV-1IIIB induces c-Src and Pyk2 activation in Jurkat C, within 15 minutes of infection. Non-targeting c-Src Pyk2 Non-targeting c-Src Pyk2 Western Blot siRNA siRNA Empty vector c-Src/β-actin T = 15 min T = 0 min Jurkat *** P < 0.001 Pyk2/β-actin Wild type c-Src Dominant negative c-Src 130 95 A. Total phosphotyrosine proteins 72 55 Conclusions 36 Hypothesis 28 The c-Src kinase inhibitor SU6656 increased HIV-1/VSV-G infection in Jurkat C and JE6-1 T-cells. Adenovirus containing a kinase inactive c-Src mutant increased HIV-1/VSV-G infection in three T-cell lines. 3. Knocking down c-Src or Pyk2 mRNA in JE6-1 cells led to an increase in HXB2 infection. Upon entering a cell, HIV-1 causes the activation of many cellular tyrosine kinases. We hypothesize that early activation of Pyk2 and c-Src are cellular responses which impede infection. B. c-src C. Pyk2 2. SU6656 inhibits c-Src activity and increases VSV-G/HIV-1 infection in two Jurkat T-cell lines. * P < 0.05 ** P < 0.005 A) Luciferase Activity B) Kinase Assay Methods Jurkat C Jurkat C JE6-1 ** * 100 10000 10000 Anticipated Significance * 1. Immunoprecipitate phosphoproteins Jurkat C Relative Activity (%) RLU (units) RLU (units) 50 Discovering host kinases critical to HIV-1 replication may provide new targets for anti-viral therapy. Kinase inhibitors already exist for various cancer treatments, these could be translated as viable options to treat HIV-1 infection. Better understanding of how viruses manipulate host signaling cascades. Cell lysis Immunoprecipitate phosphoproteins with ptyr antibody Infect with HIV-1IIIB for 15 min SDS-PAGE and Western blot 0 0 0 4. siRNA knockdown VSV-G VSV-G DMSO DMSO DMSO SU6656 20uM SU6656 10uM SU6656 10uM SU6656 20uM Infect with HXB2 for 2 days SU6656 10uM SU6656 20uM JE6-1 2. Drug inhibition of c-Src Luciferase assay 3. Dominant negative (DN) c-Src causes a higher infection of VSV-G/HIV-1. JE6-1 Jurkat C Measure protein knockdown by Western blot densitometry Kinase assay Transfect CD4+ T-cells with siRNA for 24 hr (cationic lipid method) * P < 0.05 B) Luciferase Activity A) FACS Infect with VSV-G/HIV-1 for 2 days Wash with PBS Pre-incubate with SU6656 for 1 hr * Luciferase assay 100 * P = 0.06 Acknowledgments 3. Adenovirus gene transduction 500 10000 5000 Infect with VSV-G/HIV-1 for 2 days RLU (units/200,000 cells) JE6-1 Hut78 Kit225 % of cells YFP + 50 This work was supported by the Ontario HIV Treatment Network. Support was partially provided by Vanier CGS D and NSERC CGS M scholarships awarded to Stephen McCarthy. We thank YuliaKatsman and Amanda Harrison Wong for their assistance with experiments. Luciferase assay Measure transduction efficiency (FACS) Administer adenovirus containing eYFP and Src gene for 24hr 0 0 0 0 JE6-1 Hut78 Kit225 JE6-1 Hut78 Kit225

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