Wu J, Amandoron E, Li X, Wainberg M A, Parniak M A
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1993 May 15;268(14):9980-5.
A series of monoclonal antibodies against p51/p66 human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) were prepared by immunizing mice with the native enzyme immobilized on nitrocellulose. One of these antibodies, designated 1E8, was found to inhibit both RNA-dependent and DNA-dependent polymerase activities of RT but had no effect on the RNase H activity of the enzyme. This inhibition was noncompetitive with respect to primer/template and competitive with respect to deoxynucleoside triphosphate (dNTP). The extent of 1E8 inhibition of RT polymerase activity decreased with increasing concentrations of dNTP in the incubation but was not affected by changes in primer/template concentration. 1E8 bound equally well in solution to both free RT and to the RT-primer/template complex. However, binding to the latter was significantly reduced by the addition of increasing concentrations of dNTP. The ability of dNTP to inhibit the interaction of 1E8 with the RT-primer/template complex was dependent on the identity of the homopolymeric primer/template used; only that dNTP complementary to the template was effective in this respect. 1E8 bound to the p51/p66 reverse transcriptase heterodimer in solution and reacted with both p51 and p66 subunits of reverse transcriptase on Western blots. The antibody is therefore presumed to recognize a linear surface epitope on the enzyme. 1E8 was found to specifically recognize a peptide with the sequence KKDSTKWRK. This sequence corresponds to residues 65-73 of HIV-1 reverse transcriptase, a region identified as highly antigenic by several computer algorithms. Two mutations within this sequence have been identified with resistance to 3'-azido,3'-deoxythymidine. We conclude that residues 65-73 of HIV-1 reverse transcriptase may be at or near the polymerase active site of the enzyme, and may form part of the deoxynucleoside triphosphate binding domain of the enzyme.
通过用固定在硝酸纤维素上的天然酶免疫小鼠，制备了一系列针对p51/p66人免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的单克隆抗体。其中一种抗体，命名为1E8，被发现能抑制RT的RNA依赖性和DNA依赖性聚合酶活性，但对该酶的核糖核酸酶H活性没有影响。这种抑制作用在引物/模板方面是非竞争性的，而在脱氧核苷三磷酸（dNTP）方面是竞争性的。随着孵育中dNTP浓度的增加，1E8对RT聚合酶活性的抑制程度降低，但不受引物/模板浓度变化的影响。1E8在溶液中与游离RT以及RT-引物/模板复合物的结合效果相同。然而，随着dNTP浓度的增加，与后者的结合显著减少。dNTP抑制1E8与RT-引物/模板复合物相互作用的能力取决于所使用的同聚物引物/模板的同一性；在这方面，只有与模板互补的dNTP是有效的。1E8在溶液中与p51/p66逆转录酶异二聚体结合，并在蛋白质印迹中与逆转录酶的p51和p66亚基发生反应。因此，推测该抗体识别该酶上的线性表面表位。发现1E8能特异性识别序列为KKDSTKWRK的肽。该序列对应于HIV-1逆转录酶的65-73位残基，这是一个被几种计算机算法鉴定为高度抗原性的区域。已在该序列内鉴定出两个对3'-叠氮基-3'-脱氧胸苷具有抗性的突变。我们得出结论，HIV-1逆转录酶的65-73位残基可能位于该酶的聚合酶活性位点处或附近，并且可能构成该酶的脱氧核苷三磷酸结合结构域的一部分。
