Harris D, Engelman A
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
J Biol Chem. 2000 Dec 15;275(50):39671-7. doi: 10.1074/jbc.M002626200.
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.
逆转录病毒整合由病毒前整合复合物（PIC）介导，用高盐处理的1型人类免疫缺陷病毒（HIV-1）PIC会丧失其体外整合活性。自身整合屏障因子（BAF）是一种宿主蛋白，可有效恢复PIC活性，但BAF参与HIV-1整合的机制仍 largely未知。在这里，我们开发了一种凝胶迁移试验来研究BAF与DNA的结合，并分析了14种含有保守残基替代的突变蛋白的结合和PIC重组活性。虽然野生型BAF能有效结合双链DNA，但未检测到其与单链DNA、RNA或RNA/DNA杂交体的结合，这表明BAF在逆转录过程中相对较晚才与逆转录病毒cDNA结合。虽然一些BAF突变蛋白能有效结合DNA，但其他一些则存在结合缺陷。能有效结合DNA的突变体可重组HIV-1整合，尽管在一种情况下结合仅为野生型BAF的0.2%。虽然错误折叠的突变体不能重组整合，但一个结构完整但缺乏DNA结合能力的突变体在高BAF浓度下显示出部分活性。因此，我们得出结论，BAF蛋白结构及其DNA结合活性在体外重组HIV-1整合过程中均发挥作用。
