Woerner A M, Marcus-Sekura C J
Division of Viral Products, FDA, Bethesda, MD 20892.
Nucleic Acids Res. 1993 Jul 25;21(15):3507-11. doi: 10.1093/nar/21.15.3507.
The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.
人类免疫缺陷病毒1型（HIV-1）的整合酶（IN）蛋白催化从病毒长末端重复序列（LTR）中特异性切割2个碱基，但它与DNA结合时几乎没有DNA序列特异性。我们之前已经证明，IN的C端一半（氨基酸154 - 288）具有一个DNA结合结构域。为了进一步表征该区域，构建了一系列在大肠杆菌中表达截短形式IN作为N端融合蛋白的克隆，并通过蛋白质印迹法进行分析。包含氨基酸1 - 263、1 - 248和170 - 288的蛋白质保留了与DNA结合的能力，而包含氨基酸1 - 180的蛋白质未显示出可检测到的DNA结合。这确定了氨基酸180 - 248内包含的一个DNA结合结构域。该区域包含一组9个赖氨酸和精氨酸残基，每个残基间隔2 - 4个氨基酸（KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK），跨越氨基酸211 - 244，在所有HIV-1分离株中都是保守的。一个表达带有16个氨基酸C端融合的全长IN的克隆与一个具有游离C端的克隆蛋白结合DNA的能力相当，并且一种识别氨基酸264 - 279内表位的IN特异性单克隆抗体无法阻断DNA结合，支持了结合所需区域位于氨基酸264上游的证据。
