Both the structure and DNA binding function of the barrier-to-autointegration factor contribute to reconstitution of HIV type 1 integration in vitro.
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.
Harris
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D
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Engelman
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A
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D011485:Protein Binding; D017510:Protein Folding; D012313:RNA / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
C501959:BANF1 protein, human; C501960:Banf1 protein, mouse; C501961:Banf1 protein, rat; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012313:RNA; D004247:DNA