Pandey Krishan K, Bera Sibes, Zahm Jacob, Vora Ajaykumar, Stillmock Kara, Hazuda Daria, Grandgenett Duane P
Institute for Molecular Virology, Saint Louis University Health Sciences Center, St Louis, MO 63110, USA.
J Virol. 2007 Nov;81(22):12189-99. doi: 10.1128/JVI.02863-06. Epub 2007 Sep 5.
Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (~15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3'-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3'-OH processing. It follows that the IN-viral DNA complex is "trapped" by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.
1型人类免疫缺陷病毒（HIV-1）整合酶（IN）将病毒DNA基因组插入宿主染色体。在此，我们通过天然琼脂糖凝胶电泳，使用带有平端病毒DNA底物的重组IN，鉴定出了突触复合体（SC），这是整合途径中的一种瞬时早期中间体。SC由两个通过IN非共价并列的供体末端组成。SC内的DNA末端加工极少（约15%）。SC以时间依赖性方式与靶DNA结合，并进展为链转移复合体（STC），即协同整合的核蛋白产物。在STC中，两个病毒DNA末端共价连接到靶上，并与IN保持结合。二酮酸抑制剂及其类似物通过在体内阻止整合来有效抑制HIV-1复制。链转移抑制剂L-870,810、L-870,812和L-841,411在低纳摩尔浓度下能有效抑制病毒DNA供体在体外的协同整合。这些抑制剂以浓度依赖性方式与SC内的IN结合，从而阻断与靶DNA的对接，进而阻止STC的形成。尽管3'-OH凹陷的供体能高效形成STC，但用该底物进行的反应对抑制剂的存在表现出明显抗性，需要显著更高的浓度才能有效抑制所有链转移产物。这些结果表明，抑制剂与SC的结合发生在3'-OH加工之前、期间或之后立即发生。由此可见，IN-病毒DNA复合体在细胞质预整合复合体内通过一个瞬时中间体被链转移抑制剂“捕获”。
