Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction
Open Access
- 15 April 2003
- journal article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 77 (8) , 4685-4694
- https://doi.org/10.1128/jvi.77.8.4685-4694.2003
Abstract
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 α-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.Keywords
This publication has 71 references indexed in Scilit:
- Kinetics of Human Immunodeficiency Virus Type 1 (HIV) DNA Integration in Acutely Infected Cells as Determined Using a Novel Assay for Detection of Integrated HIV DNAJournal of Virology, 2001
- HIV DNA integration during cell-to-cell transmission of infection: evidence for partially integrated DNA structures in acutely infected cellsArchiv für die gesamte Virusforschung, 2001
- Characterization of the Nuclear Import Pathway for HIV-1 IntegraseJournal of Biological Chemistry, 2001
- The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brainNature Biotechnology, 2001
- Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 VprJournal of Virology, 2001
- DNA-Dependent Protein Kinase Is Not Required for Efficient Lentivirus IntegrationJournal of Virology, 2000
- In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral VectorScience, 1996
- Kinetic Analysis of HIV-1 Early Replicative Steps in a Coculture SystemAIDS Research and Human Retroviruses, 1994
- Restriction endonuclease cleavage of linear and closed circular murine leukemia viral DNAS: Discovery of a smaller circular formCell, 1979
- Mapping unintegrated avian sarcoma virus DNA: Termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNACell, 1978