Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
J Biol Chem. 2011 Jan 28;286(4):3018-32. doi: 10.1074/jbc.M110.175018. Epub 2010 Nov 5.
A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2(FLAG), produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2(FLAG) particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2(FLAG) particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.
丙型肝炎病毒 (HCV) 颗粒的一个标志是它们与宿主细胞脂质的关联，尤其是脂蛋白成分。人们认为，这种特性解释了病毒颗粒的低密度和它们的巨大异质性。然而，感染性病毒粒子的组成及其生化和形态特性在很大程度上是未知的。我们开发了一种系统，其中包膜糖蛋白 E2 的 N 端被 FLAG 表位标记。这种病毒被命名为 Jc1E2(FLAG)，产生与野生型相当的感染滴度，并允许亲和纯化病毒粒子，然后对其蛋白质和脂质组成进行分析。通过使用质谱法，我们发现 Jc1E2(FLAG)颗粒的脂质组成类似于极低密度和低密度脂蛋白，其中胆固醇酯占总 HCV 脂质的近一半。因此，HCV 颗粒具有独特的脂质组成，与迄今为止分析的所有其他病毒以及产生 HCV 的人类肝细胞非常不同。通过电子显微镜 (EM)，我们发现纯化的 Jc1E2(FLAG)颗粒是异质的，主要是球形结构，平均直径约为 73nm。重要的是，通过免疫电镜评估，我们发现大多数含有 E2 的颗粒表面还含有 apoE。总之，我们描述了一种快速有效的生产大量亲和纯化 HCV 的系统，允许对感染性病毒粒子进行全面分析，包括确定其脂质组成。
