Ehrlich L S, Agresta B E, Carter C A
Department of Microbiology, State University of New York, Stony Brook 11794.
J Virol. 1992 Aug;66(8):4874-83. doi: 10.1128/JVI.66.8.4874-4883.1992.
The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures. The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis. Chemical cross-linking of CA molecules was observed with several homobifunctional reagents. Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers. Oligomers as large as dodecamers were detected in native polyacrylamide gels. These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization. Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure. This region was mapped to the central portion of the protein. The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH. The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly.
在大肠杆菌中表达并纯化至同质性大于90%的1型人类免疫缺陷病毒（HIV）的衣壳蛋白（CA）（p24），被用于体外组装研究，并探究衣壳结构形成过程中相互作用的本质。通过凝胶过滤柱色谱法、蔗糖沉降法和非变性凝胶电泳进行分子大小测量，结果表明该蛋白以二聚体和寡聚体形式存在。用几种同型双功能试剂观察到了CA分子的化学交联。寡聚体大小取决于交联剂浓度，并呈现出一种非随机模式，其中二聚体和四聚体比三聚体和五聚体更为丰富。在天然聚丙烯酰胺凝胶中检测到了高达十二聚体的寡聚体。这些寡聚体在高离子强度溶液或存在非离子去污剂的情况下是稳定的，这表明寡聚体稳定化涉及强相互作用。有限的胰蛋白酶消化将假定的十二聚体转化为八聚体，这表明参与CA蛋白多聚化的一个区域在结构中是暴露的。该区域被定位到蛋白的中央部分。重组CA蛋白在体外组装成长棒状结构，并通过改变离子强度和pH值解聚成小的不规则球体。纯化的1型HIV CA蛋白在体外可被诱导组装和解聚，这一观察结果提示了一种识别HIV病毒核心组装中可能控制机制的方法。
