Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.
The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. 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) or 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+. Both metal ions were equally effective for the circular half-site reaction 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 were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). 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 recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.
Goodarzi
G
G
St. Louis University Health Science Center, Institute for Molecular Virology, Missouri 63110, USA.
Im
G J
GJ
Brackmann
K
K
Grandgenett
D
D
D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004251:DNA Transposable Elements; D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D003902:Detergents; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D019426:Integrases; D007700:Kinetics; D010957:Plasmids; D015183:Restriction Mapping; D017384:Sequence Deletion; D014771:Virion / Q000235:genetics / Q000502:physiology; D016662:Virus Integration
D004251:DNA Transposable Elements; D004270:DNA, Circular; D004279:DNA, Viral; D003902:Detergents; D004254:DNA Nucleotidyltransferases; D019426:Integrases