Gilead Sciences, Foster City, California 94404, United States.
Biochemistry. 2012 Jun 5;51(22):4416-28. doi: 10.1021/bi300052h. Epub 2012 May 18.
Human immunodeficiency virus-1 (HIV-1) capsid protein (CA) has become a target of antiviral drug design in recent years. The recognition that binding of small molecules to the CA protein can result in the perturbation of capsid assembly or disassembly has led to mathematical modeling of the process. Although a number of capsid assembly models have been developed using biophysical parameters of the CA protein obtained experimentally, there is currently no model of CA polymerization that can be practically used to analyze in vitro CA polymerization data to facilitate drug discovery. Herein, we describe an equilibrium model of CA polymerization for the kinetic analysis of in vitro assembly of CA into polymer tubes. This new mathematical model has been used to assess whether a triangular trimer of dimers rather than a hexagonal hexamer can be the basic capsomere building block of CA polymer. The model allowed us to quantify for the first time the affinity for each of the four crucial interfaces involved in the polymerization process and indicated that the trimerization of CA dimers is a relatively slow step in CA polymerization in vitro. For wild-type CA, these four interfaces include the interface between two monomers of a CA dimer (K(D) = 6.6 μM), the interface between any two dimers within a CA trimer of dimers (K(D) = 32 nM), and two types of interfaces between neighboring trimers of dimers, either within the same ring around the perimeter of the polymer tube (K(D) = 438 nM) or from two adjacent rings (K(D) = 147 nM). A comparative analysis of the interface dissociation constants between wild-type and two mutant CA proteins, cross-linked hexamer (A14C/E45C/W184A/M185A) and A14C/E45C, yielded results that are consistent with the trimer of dimers with a triangular geometry being the capsomere building block involved in CA polymer growth. This work provides additional insights into the mechanism of HIV-1 CA assembly and may prove useful in elucidating how small molecule CA binding agents may disturb this essential step in the HIV-1 life cycle.
人类免疫缺陷病毒 1（HIV-1）衣壳蛋白（CA）近年来已成为抗病毒药物设计的靶标。人们认识到，小分子与 CA 蛋白的结合可导致衣壳组装或解体的扰动，这导致了该过程的数学建模。尽管已经使用从 CA 蛋白获得的实验生物物理参数开发了许多衣壳组装模型，但目前尚无可实际用于分析体外 CA 聚合数据以促进药物发现的 CA 聚合模型。在此，我们描述了一种 CA 聚合的平衡模型，用于分析 CA 体外组装成聚合物管的动力学。该新数学模型已用于评估三角形三聚体二聚体而不是六边形六聚体是否可以作为 CA 聚合物的基本衣壳结构基元。该模型首次使我们能够量化参与聚合过程的四个关键界面中的每一个界面的亲和力，并表明 CA 二聚体的三聚化是 CA 体外聚合的相对缓慢步骤。对于野生型 CA，这四个界面包括 CA 二聚体中两个单体之间的界面（K(D) = 6.6 μM），CA 三聚体中二聚体之间的任何两个界面（K(D) = 32 nM），以及两种类型的二聚体相邻三聚体之间的界面，要么在聚合物管周界周围的同一环内（K(D) = 438 nM），要么在两个相邻环内（K(D) = 147 nM）。野生型和两种突变体 CA 蛋白（交联六聚体（A14C/E45C/W184A/M185A）和 A14C/E45C）之间界面离解常数的比较分析产生的结果与具有三角形几何形状的三聚体二聚体作为涉及 CA 聚合物生长的衣壳结构基元一致。这项工作提供了对 HIV-1 CA 组装机制的更多了解，并可能有助于阐明小分子 CA 结合剂如何干扰 HIV-1 生命周期中的这一关键步骤。
