Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features
Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features
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DOI：
10.1128/jvi.79.17.11353-11365.2005
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发表时间：
2005-09-01
影响因子：
5.4
通讯作者：
Lavergne, JP
中科院分区：
文献类型：
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作者：
Boulant, S;Vanbelle, C;Lavergne, JP
丙型肝炎病毒核衣壳的核心蛋白与病毒RNA一起，由参与RNA结合和同源寡聚的不同结构域组成。在大肠杆菌中表达了丙型肝炎病毒核心蛋白1-169(C(丙型肝炎病毒)169)及其N末端1-117位(C(丙型肝炎病毒)117)，并对其进行了纯化，得到了适合生化和生物物理特性的均一蛋白。用分析离心法、圆二色谱、本征荧光测量和有限蛋白水解法研究了C(丙型肝炎病毒)169和C(丙型肝炎病毒)17蛋白的整体构象和寡聚体性质。总之，我们的结果表明，核心蛋白(C(丙型肝炎病毒)169)在没有洗涤剂的情况下表现为膜蛋白，并形成高相对分子质量的异质可溶胶束状聚集体。相反，在温和的洗涤剂存在下，它表现为一种可溶的、折叠良好的非共价二聚体。与观察到的丙型肝炎病毒黄病毒核心蛋白相似，丙型肝炎病毒核心蛋白主要由α-螺旋组成(50%)。相反，C(丙型肝炎病毒)117在没有洗涤剂的情况下是可溶的和单分散的，但是展开的。似乎高碱性结构域从位置2到117(2-117结构域)的折叠取决于117-169疏水结构域的存在，该结构域包含确保核心与细胞膜结合的结构决定因素。最后，我们的发现为进一步研究分离的核心蛋白以及尝试在体外重建核衣壳颗粒提供了有价值的信息。
The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C(HCV)169) and its N-terminal region from positions 1 to 117 (C(HCV)117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C(HCV)169 and C(HCV)17 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C(HCV)169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of a-helices (50%). In contrast; C(HCV)117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.