Journal of Biological Chemistry
Volume 275, Issue 50, 15 December 2000, Pages 39671-39677
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DNA: REPLICATION REPAIR AND RECOMBINATION
Both the Structure and DNA Binding Function of the Barrier-to-Autointegration Factor Contribute to Reconstitution of HIV Type 1 Integration in Vitro *

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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 vitrointegration 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.

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Published, JBC Papers in Press, September 25, 2000, DOI 10.1074/jbc.M002626200

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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.