Baumert T F, Ito S, Wong D T, Liang T J
Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Virol. 1998 May;72(5):3827-36. doi: 10.1128/JVI.72.5.3827-3836.1998.
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.
丙型肝炎病毒（HCV）是全球慢性肝炎的主要病因。HCV研究受到以下因素的阻碍：受感染个体中病毒颗粒水平较低、无法在培养细胞中高效繁殖该病毒以及缺乏便利的动物模型。由于这些障碍，病毒的结构及其组装所需条件均未得到明确界定。在本报告中，我们描述了一种利用含有HCV结构蛋白cDNA的重组杆状病毒在昆虫细胞中生产和纯化HCV样颗粒的模型。在昆虫细胞中，表达的HCV结构蛋白在大的细胞质池中组装成包膜病毒样颗粒（直径40至60纳米），这些池大概源自内质网。通过氯化铯和蔗糖梯度离心对病毒样颗粒进行生物物理表征，结果显示其生物物理特性与从受感染人类中分离出的假定病毒粒子相似。结果表明，不含p7的HCV核心蛋白和包膜蛋白足以形成病毒颗粒。对颗粒相关核酸的分析表明，与非HCV转录本相比，HCV RNA被选择性地整合到颗粒中。在昆虫细胞中合成HCV样颗粒可能为确定HCV颗粒组装的结构要求以及研究病毒基因组包装和病毒-宿主相互作用提供重要工具。所描述的系统也可能代表一种潜在的疫苗开发方法。
