DNA-dependent protein kinase is not required for efficient lentivirus integration

How DNA is repaired after retrovirus integration is not well understood. DN A-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 retrov iral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Sk alka, Science 284:644-647, 1999). Reduced transduction efficiency and incre ased cell death by apoptosis were observed upon retrovirus infection of cul tured scid cells. We have used a human immunodeficiency virus (HM type I (H IV-I)-derived lentivirus vector system to further investigate the role of D NA-PK during integration. We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are defi cient 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 lentiv irus transduction was obtained, excluding an essential role for DNA-PK in l entivirus integration. Likewise, the efficiency of transduction of HIV-deri ved vectors in scid mouse brain was as efficient as that in control mice, w ithout 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 tite rs (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and sub sequent 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 c ontrol 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 pl ay an important role during retrovirus integration is poly(ADP-ribose) poly merase (PARP). However, no inhibition of lentivirus vector-mediated transdu ction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, w as observed. In conclusion, DNA-PK and PARP are not essential for lentiviru s integration.