Impact of the central polypurine tract on the kinetics of human immunodeficiency virus type 1 vector transduction.
Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor alpha-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration.
Van Maele
Bénédicte
B
Rega Institute for Medical Research, Leuven, Flanders, Belgium.
De Rijck
Jan
J
De Clercq
Erik
E
Debyser
Zeger
Z
D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016325:HIV Long Terminal Repeat / Q000235:genetics / Q000502:physiology; D015497:HIV-1 / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011533:Proviruses; D011687:Purines / Q000378:metabolism*; D014161:Transduction, Genetic; D016662:Virus Integration; D014779:Virus Replication
D004279:DNA, Viral; D011687:Purines