title: DNA-Dependent protein kinase is not required for efficient lentivirus integration.
abstract: How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.
authors.affiliation: Laboratory for Experimental Neurosurgery and Neuroanatomy, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: V
authors.initials: V
authors.lastname: Baekelandt
authors.forename: A
authors.initials: A
authors.lastname: Claeys
authors.forename: P
authors.initials: P
authors.lastname: Cherepanov
authors.forename: E
authors.initials: E
authors.lastname: De Clercq
authors.forename: B
authors.initials: B
authors.lastname: De Strooper
authors.forename: B
authors.initials: B
authors.lastname: Nuttin
authors.forename: Z
authors.initials: Z
authors.lastname: Debyser
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000502:physiology; D022761:Gene Transfer, Horizontal; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D009687:Nuclear Proteins / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D016662:Virus Integration
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen