IDENTIFICATION OF AMINO-ACID-RESIDUES CRITICAL FOR ENDONUCLEASE AND INTEGRATION ACTIVITIES OF HIV-1 IN PROTEIN INVITRO
IDENTIFICATION OF AMINO-ACID-RESIDUES CRITICAL FOR ENDONUCLEASE AND INTEGRATION ACTIVITIES OF HIV-1 IN PROTEIN INVITRO
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DOI：
10.1016/0042-6822(92)90499-f
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发表时间：
1992-06-01
期刊：
影响因子：
3.7
通讯作者：
MOUS, J
中科院分区：
文献类型：
--
作者：
DRELICH, M;WILHELM, R;MOUS, J
在大肠杆菌中表达了带有六聚组氨酸尾的HIV-IN蛋白，并通过一步镍螯合亲和层析法纯化。纯化的IN蛋白的特征在于其在体外的内切酶和整合酶的性质。在2-5 mMMg ~（2+）或Ca ~（2+）存在下观察到HIV U 5LTR末端的特异性切割和整合。在存在2 mMMn 2+的情况下，在额外位点发生切割和整合，表明特异性降低。在体外检测了突变IN蛋白的性质。从N-末端缺失39个氨基酸和从C-末端缺失至少25个残基会损害IN介导的切割和整合活性。定点突变实验的结果表明，IN功能的关键残基是高度保守的。保守残基Asp 64、Pro 109 Asp 116和Glu 152的突变在体外对IN功能产生不利影响。非保守残基Gly 189和Thr 112的突变没有影响。保守的Thr 115突变为Ala导致几乎完全丧失Mg 2+依赖的整合活性，但仅部分影响IN的内切核酸切割活性。这些结果表明，并不是所有的保守残基参与核酸内切酶切割和整合活动的HIV-IN。
HIV-IN protein, tagged with a hexahistidine tail was expressed inEscherichia coliand purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase propertiesin vitro. Specific cleavage and integration of HIV U5LTR ends were observed in the presence of 2–5 mMMg2+or Ca2+. In the presence of 2 mMMn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examinedin vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109Asp116, and Glu152adversely affected IN functionin vitro. Mutations of nonconserved residues Gly189and Thr112had no effect. Mutation of a conserved Thr115to Ala caused a near complete loss of Mg2+-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.