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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: June 1st, 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.

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*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

© 2014 by Lippincott Williams & Wilkins