title: Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors.
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 about 30 nM IN (20 min at 37 degrees 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 about 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 (about 125 microg/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.
authors.affiliation: Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri 63110, USA.
authors.forename: Sapna
authors.initials: S
authors.lastname: Sinha
authors.forename: Michael H
authors.initials: MH
authors.lastname: Pursley
authors.forename: Duane P
authors.initials: DP
authors.lastname: Grandgenett
mesh_terms: D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D019427:HIV Integrase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D013997:Time Factors; D016662:Virus Integration; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D011994:Recombinant Proteins; D019427:HIV Integrase