Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors
- 1 April 2002
- journal article
- research article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 76 (7), 3105-3113
- https://doi.org/10.1128/jvi.76.7.3105-3113.2002
Abstract
Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At ∼30 nM IN (20 min at 37°C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of ∼70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (∼125 μg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.Keywords
This publication has 30 references indexed in Scilit:
- HIV Integrase, a Brief Overview from Chemistry to TherapeuticsJournal of Biological Chemistry, 2001
- DNase Protection Analysis of Retrovirus Integrase at the Viral DNA Ends for Full-Site Integration In VitroJournal of Virology, 2001
- Base-Pair Substitutions in Avian Sarcoma Virus U5 and U3 Long Terminal Repeat Sequences Alter the Process of DNA Integration In VitroJournal of Virology, 2001
- Photo-Cross-Linking Studies Suggest a Model for the Architecture of an Active Human Immunodeficiency Virus Type 1 Integrase−DNA ComplexBiochemistry, 1998
- Zn2+ Promotes the Self-Association of Human Immunodeficiency Virus Type-1 Integrase in VitroBiochemistry, 1997
- A Soluble Active Mutant of HIV-1 IntegraseJournal of Biological Chemistry, 1996
- The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitroCell, 1990
- The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitroCell, 1990
- GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coliNature, 1989
- Correct integration of retroviral DNA in vitroCell, 1987