Kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro
Kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro
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DOI：
10.1128/jvi.76.14.6900-6908.2002
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发表时间：
2002-07-01
影响因子：
5.4
通讯作者：
Prevelige, PE
中科院分区：
文献类型：
--
作者：
Lanman, J;Sexton, J;Prevelige, PE
人类免疫缺陷病毒1型（HIV-1）衣壳蛋白（CA）在病毒粒子的组装和成熟中起着至关重要的作用。最近的许多研究都集中在CA的可溶形式或体外CA组装的聚合物最终产物上。特别是CA聚合物，已被用于研究CA-CA相互作用，因为它是病毒粒子核心内CA相互作用的良好模型。然而，对体外CA组装过程的分析可以对CA-CA相互作用和核心组装机制产生有价值的见解。我们在这里描述了一种分析CA装配动力学的方法，其中通过使用浊度来监测装配的进展。在pH 7.0时，将分离的CA结构域（即N或C结构域）中的任何一个加入到组装反应中，通过竞争与全长CA蛋白的结合，导致组装速率降低。在pH 8.0时，分离的C结构域的加入对CA的组装有类似的抑制作用。然而，在pH 8.0时，分离的N结构域对CA的组装速度没有影响，但当与C结构域混合时，它减轻了C结构域的抑制作用。这些数据为ph敏感的同型N结构域相互作用以及N-和c -结构域相互作用提供了生化证据。
The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion. Numerous recent studies have focused on either the soluble form of CA or the polymer end product of in vitro CA assembly. The CA polymer, in particular, has been used to study CA-CA interactions because it is a good model for the CA interactions within the virion core. However, analysis of the process of in vitro CA assembly can yield valuable insights into CA-CA interactions and the mechanism of core assembly. We describe here a method for the analysis of CA assembly kinetics wherein the progress of assembly is monitored by using turbidity. At pH 7.0 the addition of either of the isolated CA domains (i.e., the N or the C domain) to an assembly reaction caused a decrease in the assembly rate by competing for binding to the full-length CA protein. At pH 8.0 the addition of the isolated C domain had a similar inhibitory affect on CA assembly. However, at pH 8.0 the isolated N domain had no affect on the rate of CA assembly but, when mixed with the C domain, it alleviated the C-domain inhibition. These data provide biochemical evidence for a pH-sensitive homotypic N-domain interaction, as well as for an N- and C-domain interaction.