Sherman P A, Fyfe J A
Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, NC 27709.
Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119-23. doi: 10.1073/pnas.87.13.5119.
The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.
人类免疫缺陷病毒（HIV）整合蛋白是选择性抗病毒治疗的一个潜在靶点，它在大肠杆菌中表达。纯化后的蛋白未检测到污染性核酸内切酶，能选择性切割双链DNA寡核苷酸，这些寡核苷酸模拟线性HIV DNA的U3和U5末端。U5正链和U3负链的3'端都去除了两个核苷酸；在这两种情况下，切割都发生在保守的CA二核苷酸相邻处。该反应依赖金属离子，相对于Mg2+，更偏好Mn2+。HIV U5互补（负）链底物、模拟禽逆转录病毒U3末端的类似底物以及保守CA二核苷酸被TA二核苷酸取代的HIV U5底物均未发生切割，进一步证明了反应的选择性。这种整合蛋白介导的切割反应预计会作为逆转录病毒生命周期中整合事件的一部分发生，在该过程中，病毒RNA基因组的双链DNA拷贝会插入宿主细胞DNA中。
