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: firstname.lastname@example.org).
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.
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Received December 31, 2013
Accepted December 31, 2013