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J. Biol. Chem., Vol. 275, Issue 50, 39671-39677, December 15, 2000
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From the Department of Cancer Immunology and AIDS, Dana-Farber
Cancer Institute and the Department of Pathology, Harvard Medical
School, Boston, Massachusetts 02115
Retroviral integration is mediated by viral
preintegration complexes (PICs), and human immunodeficiency virus type
1 (HIV-1) PICs treated with high salt lose their in vitro
integration activity. Barrier-to-autointegration factor (BAF) is a host
protein that efficiently restores PIC activity, but the mechanism(s) by
which BAF participates in HIV-1 integration remains largely unknown. Here we developed a gel shift assay to study BAF DNA binding, and
analyzed 14 mutant proteins containing substitutions of conserved residues for binding and PIC reconstitution activities. Although wild-type BAF efficiently bound double-stranded DNA, binding to single-stranded DNA, RNA, or an RNA/DNA hybrid was not detected, suggesting that BAF associates with retroviral cDNA relatively late
during reverse transcription. Although some of the BAF mutant proteins
efficiently bound DNA, others were defective for binding. Mutants that
bound DNA efficiently reconstituted HIV-1 integration, even though in
one case binding was just 0.2% of wild-type BAF. Although misfolded
mutants did not reconstitute integration, a structurally intact DNA
binding-defective mutant displayed partial activity at high BAF
concentration. We therefore conclude that both BAF protein structure
and its DNA binding activity play roles in reconstituting HIV-1
integration in vitro.
Both the Structure and DNA Binding Function of the
Barrier-to-Autointegration Factor Contribute to Reconstitution of
HIV Type 1 Integration in Vitro*
*
This work was supported by National Institutes of Health
Grant AI39394, by funds from the G. Harold and Lelia Y. Mathers
Foundation, and by a gift from the Friends 10.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cancer
Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-4361; Fax: 617-632-3113; E-mail: alan_engelman@dfci.harvard.edu.
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