Nielsen Søren U, Bassendine Margaret F, Burt Alastair D, Martin Caroline, Pumeechockchai Wanna, Toms Geoffrey L
Liver Research Group, School of Clinical Medical Sciences, University of Newcastle, England.
J Virol. 2006 Mar;80(5):2418-28. doi: 10.1128/JVI.80.5.2418-2428.2006.
Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, < or =1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.
丙型肝炎病毒（HCV）RNA在持续感染患者的血液中以脂质病毒颗粒（LVP）的形式循环，这些颗粒密度不均一，与宿主脂蛋白和抗体相关。这些病毒-宿主复合物在分级分离时的变异性和不稳定性阻碍了我们对LVP结构的理解以及对HCV病毒粒子物理化学性质的确定。在本研究中，使用三种分级分离技术，即溴化钠梯度、等渗碘克沙醇和蔗糖梯度离心法，对一名抗体阴性免疫缺陷患者的HCV进行了分析。结果表明，碘克沙醇梯度最能保留宿主脂蛋白-病毒复合物，所有HCV RNA均在密度低于1.13 g/ml处被发现，大多数处于低密度，即≤1.08 g/ml。用抗人载脂蛋白B和载脂蛋白E的多克隆抗体进行免疫沉淀，分别沉淀了91.8%和95.0%的低密度HCV，这表明宿主脂蛋白在类似于极低密度脂蛋白（VLDL）的颗粒中与HCV紧密相关。用抗糖蛋白E2的抗体进行免疫沉淀，沉淀了25%的低密度HCV，这为LVP中存在E2提供了证据。在无盐条件下用0.5%脱氧胆酸处理血清，产生的HCV密度为1.12 g/ml，沉降系数为215S。这些颗粒的直径经计算为54 nm。用0.18% NP-40处理血清，产生的HCV密度为1.18 g/ml，沉降系数为180S，直径为42 nm。免疫沉淀分析表明，用脱氧胆酸或NP-40处理血清后，载脂蛋白B仍与HCV相关，而这些去污剂可将载脂蛋白E从HCV中去除。
