Nucleic Acids Research, Vol 27, Issue 10 2202-2210, Copyright © 1999 by Oxford University Press
P Cherepanov, D Surratt, J Toelen, W Pluymers, J Griffith, E De Clercq and Z Debyser
Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into
the genome of a human cell is an essential step in the viral replication
cycle. Understanding of the integration process has been facilitated by the
development of in vitro assays using specific oligonucleotides and
recombinant integrase. However, understanding of the biology of retroviral
integration will require in vitro and in vivo model systems using long DNA
substrates that mimic the HIV cDNA. We have now studied the activity of
recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA,
containing flanking regions of approximately 200 bp that represent the
intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV).
The strand transfer products of the integration reaction can be directly
visualized after separation in agarose gels by ethidium bromide staining.
The most prominent reaction product resulted from integration of one LTR
end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of
the reaction products showed them to be products of legitimate integration
preceded by correct processing of the viral LTR ends. Hotspots for
integration were detected. Electron microscopy revealed the presence of a
range of reaction products resulting from single or multiple integration
events. The binding of HIV-1 integrase to mini-HIV DNA was visualized.
Oligomers of integrase seem to induce DNA looping whereby the enzyme often
appears to be bound to the DNA substrate that adopts the structure of a
three-site synapsis that is reminiscent of the Mu phage transposase
complex.
ARTICLES
Activity of recombinant HIV-1 integrase on mini-HIV DNA
Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium and The Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.
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