title: c-SRC protein tyrosine kinase regulates early HIV-1 infection post-entry.
abstract: OBJECTIVE
  We investigated whether HIV-1 inhibition by SRC-family kinase inhibitors is through the non-receptor tyrosine kinase pp60 (c-SRC) and its binding partner, protein tyrosine kinase 2 beta (PTK2B).
  
  
  METHODS
  CD4 T-lymphocytes were infected with R5 (JR-FL) or X4 (HXB2) HIV-1. We used SRC-family kinase inhibitors or targeted siRNA knockdown of c-SRC and PTK2B, then monitored effects on the early HIV-1 lifecycle.
  
  
  METHODS
  Four SRC-family kinase inhibitors or targeted siRNA knockdown were used to reduce c-SRC or PTK2B protein expression. Activated CD4 T-lymphocytes were infected with recombinant, nef-deficient, or replication-competent infectious viruses. Knockdown experiments examined early infection by monitoring: luciferase activity, expression of host surface receptors, reverse transcriptase activity, p24 levels and qPCR of reverse transcripts, integrated HIV-1, and two-long terminal repeat (2-LTR) circles.
  
  
  RESULTS
  All SRC-family kinase inhibitors inhibited R5 and X4 HIV-1 infection. Neither c-SRC nor PTK2B siRNA knockdown had an effect on cell surface receptors (CD4, CXCR4, and CCR5) nor on reverse transcriptase activity. However, using JR-FL both decreased luciferase activity while increasing late reverse transcripts (16-fold) and 2-LTR circles (eight-fold) while also decreasing viral integration (four-fold). With HXB2, c-SRC but not PTK2B siRNA knockdown produced similar results.
  
  
  CONCLUSIONS
  Our results suggest c-SRC tyrosine kinase is a major regulator of HIV-1 infection, participating in multiple stages of infection post-entry: Reduced proviral integration with increased 2-LTR circles is reminiscent of integrase inhibitors used in combination antiretroviral therapy. Decreasing c-SRC expression and/or activity provides a new target for antiviral intervention and the potential for repurposing existing FDA-approved kinase inhibitors.
authors.affiliation: aDepartment of Laboratory Medicine and Pathobiology, University of Toronto bCentre for Innovation, Canadian Blood Services, Toronto cDepartment of Medicine, University of Toronto dDivision of Advanced Diagnostics, Infection and Immunity Group, Toronto General Research Institute, Toronto, Ontario M5G 2M1, Canada.
authors.forename: Stephen D S
authors.initials: SD
authors.lastname: McCarthy
authors.forename: Darinka
authors.initials: D
authors.lastname: Sakac
authors.forename: Anton
authors.initials: A
authors.lastname: Neschadim
authors.forename: Donald R
authors.initials: DR
authors.lastname: Branch
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002478:Cells, Cultured; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D015497:HIV-1 / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D016662:Virus Integration; D014779:Virus Replication
chemical_list: D047428:Protein Kinase Inhibitors; D051418:Focal Adhesion Kinase 2; C103547:PTK2B protein, human; D016392:Proto-Oncogene Proteins pp60(c-src)