       Document 0109
 DOCN  M9640109
 TI    Target DNA capture by HIV-1 integration complexes.
 DT    9604
 AU    Miller MD; Bor YC; Bushman F; Infectious Disease Laboratory, Salk
       Institute for Biological; Studies, La Jolla, California 92024, USA.
 SO    Curr Biol. 1995 Sep 1;5(9):1047-56. Unique Identifier : AIDSLINE
       MED/96076268
 AB    BACKGROUND: The early steps of human immunodeficiency virus 1 (HIV-1)
       replication involve reverse transcription of the viral RNA and
       integration of the resulting cDNA into a host chromosome. The DNA
       integration step requires the integration machinery ('preintegration
       complex') to bind to the host DNA before connecting the viral and host
       DNAs. Here, we present experiments that distinguish among three possible
       pathways of target-DNA capture: repeated binding and release of target
       DNA prior to the chemical strand-transfer step; binding followed by
       facilitated diffusion along target DNA (sliding); and integration at the
       initial target-capture site. The mechanism of target-DNA capture has
       implications for the design of gene therapy methods, and influences the
       interpretation of results on the selection of integration target sites
       in vivo. RESULTS: We present new in vitro conditions that allow us to
       assemble HIV-1 integrase--the virus-encoded recombination enzyme--with a
       viral DNA and then to trap assembled complexes bound to target DNA. We
       find that complexes of integrase and viral DNA do not slide along target
       DNA substantially after binding. We confirm and extend these results by
       analyzing target capture by a hybrid protein composed of HIV-1 integrase
       linked to a sequence-specific DNA-binding domain. We find that the
       integrase domain binds quickly and tightly under the above conditions,
       thereby obstructing function of the fused sequence-specific DNA-binding
       domain. We also monitor target-DNA capture by HIV-1 preintegration
       complexes purified from freshly infected cells. Partially purified
       complexes commit quickly and stably to the first target DNA added,
       whereas preintegration complexes in crude cytoplasmic extracts do not.
       The addition of extracts from uninfected cells to partially purified
       complexes blocks quick commitment. CONCLUSIONS: Under new conditions
       favorable for the analysis of target-DNA capture in vitro, HIV-1
       integrase complexes bind quickly and stably to target DNA without
       subsequent sliding. Parallel studies of preintegration complexes support
       a model in which target-site capture in vivo is reversible as a result
       of the action of cellular factors.
 DE    Binding Sites  Dimethyl Sulfoxide  DNA Nucleotidyltransferases/ISOLATION
       & PURIF/*METABOLISM  DNA, Viral/*METABOLISM  Human  HIV Long Terminal
       Repeat  HIV-1/*GENETICS/METABOLISM  Magnesium  Plasmids  Recombinant
       Fusion Proteins/METABOLISM  Support, Non-U.S. Gov't  Support, U.S.
       Gov't, P.H.S.  *Virus Integration  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

