CONCERTED INTEGRATION OF RETROVIRUS-LIKE DNA BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE

The integration of linear retrovirus DNA by the viral integrase (IN) i nto the host chromosome occurs by a concerted mechanism (full-site rea ction). IN purified from avian myeloblastosis virus and using retrovir us-like DNA restriction fragments (487 bp in length) as donors and cir cular DNA (pGEM-3) as the target can efficiently catalyze that reactio n. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) dr U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. B oth metal ions were equally effective for the circular half-site react ion involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants w ere sequenced. A total of 55% of the 87 sequenced recombinants had hos t site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specifi c duplication predominating. The other recombinants that migrated at t he linear 3.8-kbp position were mainly small deletions that were group ed into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxid e and I,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaC l). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp re combinants relative to that of the circular half-site products that we re produced; only the quantity of donor-donor versus donor-target reco mbinants was affected. The presence of Mg2+ in the preincubation mixtu res containing donor and target substrates was not necessary for the s tability of preintegration complexes on ice or at 22 degrees C. Compar isons of the avian and HIV-1 concerted integration reactions are discu ssed.