Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome
Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome
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DOI：
10.1074/jbc.m110.175018
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发表时间：
2011-01-28
影响因子：
4.8
通讯作者：
Bartenschlager, Ralf
中科院分区：
文献类型：
--
作者：
Merz, Andreas;Long, Gang;Bartenschlager, Ralf
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结合，最明显的是脂蛋白组分。据认为，这种特性解释了病毒颗粒的低密度和它们的大异质性。然而，感染性病毒粒子的组成及其生化和形态学特性在很大程度上是未知的。我们开发了一个系统，其中包膜糖蛋白E2的N-末端标记的FLAG表位。该病毒命名为Jc 1 E2（FLAG），产生的感染性滴度达到野生型水平，并允许亲和纯化病毒颗粒，分析其蛋白质和脂质组成。通过质谱分析，我们发现Jc 1 E2 FLAG颗粒的脂质组成类似于一种非常低和低密度的脂蛋白，胆固醇酯几乎占总HCV脂质的一半。因此，HCV颗粒具有独特的脂质组成，这与迄今为止分析的所有其他病毒以及产生HCV的人肝细胞非常不同。通过电子显微镜（EM），我们发现纯化的Jc 1 E2 FLAG颗粒是异质的，大部分为球形结构，平均直径约为73 nm。重要的是，大多数含E2颗粒的表面也含有apoE，如通过免疫EM所评估的。总之，我们描述了一种快速有效的系统，用于生产大量的亲和纯化的HCV，允许全面分析的感染性病毒体，包括其脂质组成的测定。
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 Jc1E2FLAG 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 Jc1E2FLAG 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.