Fromentin Rémi, Majeau Nathalie, Laliberté Gagné Marie-Eve, Boivin Annie, Duvignaud Jean-Baptiste, Leclerc Denis
Centre de Recherche en Infectiologie, Université Laval, Qué., Canada QC G1V 4G2.
Anal Biochem. 2007 Jul 1;366(1):37-45. doi: 10.1016/j.ab.2007.03.033. Epub 2007 Apr 2.
The assembly of hepatitis C virus (HCV) is not well understood. We investigated HCV nucleocapsid assembly in vitro and the role of electrostatic/hydrophobic interactions in this process. We developed a simple and rapid in vitro assay in which the progress of assembly is monitored by measuring an increase in turbidity, thereby allowing the kinetics of assembly to be determined. Assembly is performed using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from Escherichia coli. The increase in turbidity is linked to the formation of nucleocapsid-like particles (NLPs) in solution, and nucleic acids are essential to initiate nucleocapsid assembly under the experimental conditions used. The sensitivity of NLP formation to salt strongly suggests that electrostatic forces govern in vitro assembly. Mutational analysis of C1-82 demonstrated that it is the global positive charge of C1-82 rather than any specific basic residue that is important for the assembly process. Our in vitro assembly assay provides an easy and efficient means of screening for assembly inhibitors, and we have identified several inhibitory peptides that could represent a starting point for drug design.
丙型肝炎病毒（HCV）的组装过程尚未得到充分理解。我们在体外研究了HCV核衣壳的组装以及静电/疏水相互作用在此过程中的作用。我们开发了一种简单快速的体外检测方法，通过测量浊度的增加来监测组装进程，从而确定组装动力学。使用从大肠杆菌中纯化的截短型HCV核心（C1-82）进行组装，该核心包含最小组装结构域。浊度的增加与溶液中核衣壳样颗粒（NLP）的形成有关，并且在所用实验条件下，核酸对于启动核衣壳组装至关重要。NLP形成对盐的敏感性强烈表明静电力支配体外组装。对C1-82的突变分析表明，对于组装过程而言，重要的是C1-82的整体正电荷而非任何特定的碱性残基。我们的体外组装检测为筛选组装抑制剂提供了一种简便有效的方法，并且我们已经鉴定出几种抑制性肽，它们可能代表药物设计的起点。
