title: c-Src and Pyk2 protein tyrosine kinases play protective roles in early HIV-1 infection of CD4+ T-cell lines.
abstract: BACKGROUND
  During early HIV-1 infection of CD4 T-lymphocytes, many host protein tyrosine kinases become activated within minutes, including phosphoprotein pp60 (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.
authors.affiliation: *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.
authors.forename: Stephen D S
authors.initials: SD
authors.lastname: McCarthy
authors.forename: Daniel
authors.initials: D
authors.lastname: Jung
authors.forename: Darinka
authors.initials: D
authors.lastname: Sakac
authors.forename: Donald R
authors.initials: DR
authors.lastname: Branch
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000302:isolation & purification; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D010766:Phosphorylation; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; C412373:AG 1879; D004279:DNA, Viral; C028391:G protein, vesicular stomatitis virus; D007211:Indoles; D008562:Membrane Glycoproteins; D011720:Pyrazoles; D011743:Pyrimidines; C416927:SU 6656; D013449:Sulfonamides; D014759:Viral Envelope Proteins; D000081247:CSK Tyrosine-Protein Kinase; D051418:Focal Adhesion Kinase 2; C103547:PTK2B protein, human; D019061:src-Family Kinases; C000631346:CSK protein, human