Debyser Z, De Clercq E
Rega Institute for Medical Research, Leuven, Belgium.
Protein Sci. 1996 Feb;5(2):278-86. doi: 10.1002/pro.5560050212.
The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT.
1型人类免疫缺陷病毒（HIV-1）的逆转录酶（RT）由两个分别为66 kDa和51 kDa的亚基以1:1比例组成。由于二聚化是酶活性的先决条件，干扰二聚化过程可能构成一种抑制RT的替代抗病毒策略。在此，我们描述了一种基于用亚胺二甲酯对亚基进行化学交联来研究HIV-1逆转录酶二聚化状态的体外测定方法。交联导致亚基之间形成共价键，从而使交联产物能够通过变性凝胶电泳进行分离。在1至15分钟的时间进程中，分子量大于二聚体形式的交联RT产物会积累。初步证据表明，那些高分子量产物代表三聚体和四聚体，可能是高阶RT寡聚体亚基分子内交联的结果。一种对应于HIV-1 RT连接结构域中色氨酸重复基序部分的肽可抑制交联形成以及酶活性。因此，交联测定法可用于研究抑制剂对HIV-1 RT二聚化的影响。
