Institute for Molecular Virology, Saint Louis University Health Sciences Center, Saint Louis, Missouri 63104, United States.
Biochemistry. 2011 Nov 15;50(45):9788-96. doi: 10.1021/bi201247f. Epub 2011 Oct 19.
The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.
人类免疫缺陷病毒 1 型 (HIV) 突触复合物 (SC) 的组装机制尚不清楚。分子和结构研究已经确立，HIV SC 和原型泡沫病毒 (PFV) 内合体包含一个四聚整合酶 (IN)，它催化协同整合。在 1 mM EDTA 和 10 mM MgSO 4存在下纯化的 HIV IN 主要是单体。IN 有效地促进了微摩尔浓度的 3'-OH 凹陷和平齐末端 U5 长末端重复 (LTR) 寡核苷酸 (ODN) 底物 (19-42 bp) 与环状靶 DNA 的协同整合。改变 HIV IN 到 U5 DNA 表明，IN 二聚体：DNA 末端摩尔比为 1 时，协同整合最佳。随着 ODN 长度的增加，整合活性降低，从凹陷的 18/20 或 19/21 bp 开始，到 31/33 和 40/42 bp。在这些条件下，具有凹陷和平齐末端底物的 HIV 5 bp 宿主位点复制的平均保真度为 56%。在更长的 DNA（1.6 kb）上从末端开始的 U5 LTR 序列的修饰不会改变~32 bp 的 DNaseI 保护足迹，这表明 21 bp 以外的病毒序列对于 IN 结合不是必需的。结果表明，IN 与 18/20 bp 的结合不同于与 40/42 bp 的 ODN 底物的结合，用于协同整合。HIV IN 单体可能是在没有或存在链转移抑制剂的情况下尝试 IN-DNA 复合物结晶的合适候选物。
