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Ultrastructural study on the virion morphogenesis in the hepatitis C virus-infected cells

丙型肝炎病毒感染细胞病毒颗粒形态发生的超微结构研究

基本信息

批准号：
09470134
负责人：
WATANABE Shozo
金额：
$ 8.38万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470134/
关键词：
hepatitis C virus (HCV)HCV RNA HCV core particle HCV virion IMY cell immunogold electron microscopy C型肝炎ウイルス回転焼付け法 RTD-PCR 金コロイド免疫電顕法 HCVコア蛋白質

项目摘要

Hybrid cells derived from human hepatocytes and HepG2 cells (IMY cells) were transfected with entire HCV cDNA including a novel 98-nucleotide sequence donwstream from the poly(U) stretch at the 3' end of the genome, and then the cells were infected with recombinant vaccinia virus or adenovirus expressing T7 RNA polymerase. Replication of the transfected HCV genome in the cells was evidenced by detecting the cellular expression of all HCV proteins and the presence of both positive-and negative-strand HCV RNAs. In the cells, clusters of 30-35 nm electron-dense viral core-like particles at the cytoplasmic amorphous matrix area and 50-60 nm virus-like particles within dilated cisternae of the endoplasmic reticulum were observed by conventional passing through electron microscopy. Some of the latter particles showed the budding feature of a virion into the cisternae of the endoplasmic reticulum. Indirect immunogold electron microscopy using antibodies to HCV core protein and HCV envelope pr … More otein revealed that 30-35 nm particles were HCV core particles and 50-60 nm particles were HCV virions. As to the culture media of these cells, negatively stained 55 - 65 nm spherical virus-like particles with surface spike-like projections were visualized in 1.12 - 1.15 g/ml sucrose density gradient fractions which contained the highest titers of HCV RNA. Indirect immunogold electron microscopy using anti-HCV envelope antibody characterized that these particles were HCV virions, as observed specific gold labeling to antibody haloes on the surface of HCV particles, and the virus morphology was similar to that in the HCV RNA-positive human plasma previously reported by us.These findings show convincingly that HCV RNA replication and virus particle production are processed in the culture IMY cells introduced the full-length HCV cDNA by using transfection/infection protocol, and suggest that core proteins of HCV are synthesized and assembled into the core particles in the cytoplasm, and then bud into the cisternae of the endoplasmic reticulum to form the enveloped HCV virions. Less

将人肝细胞和HepG2细胞（IMY细胞）的HCV cDNA全长（包括基因组3'端poly(U)延伸下游的一个新的98个核苷酸序列）转染杂交细胞，然后用表达T7 RNA聚合酶的重组痘苗病毒或腺病毒感染细胞。通过检测所有HCV蛋白的细胞表达以及阳性和阴性链HCV rna的存在，证明了转染的HCV基因组在细胞中的复制。在细胞内，常规透射电镜观察到细胞质无定形基质区有30-35 nm的电子致密病毒核状颗粒簇，内质网扩张池内有50-60 nm的病毒样颗粒簇。后者的一些颗粒表现出病毒粒子进入内质网池的出芽特征。利用HCV核心蛋白抗体和HCV包膜蛋白的间接免疫金电镜观察，发现30 ~ 35 nm的颗粒为HCV核心颗粒，50 ~ 60 nm的颗粒为HCV病毒粒子。在含HCV RNA滴度最高的1.12 ~ 1.15 g/ml蔗糖密度梯度组中，可见55 ~ 65 nm呈阴性染色的球形病毒样颗粒，表面呈尖峰状突起。使用抗HCV包膜抗体的间接免疫金电镜观察发现，这些颗粒为HCV病毒粒子，在HCV颗粒表面观察到抗体光晕的特异性金标，病毒形态与我们之前报道的HCV rna阳性人血浆中的病毒形态相似。这些研究结果令人信服地表明，HCV RNA的复制和病毒颗粒的产生是在通过转染/感染方案引入全长HCV cDNA的培养IMY细胞中进行的，表明HCV核心蛋白在细胞质中合成并组装成核心颗粒，然后芽入内质网池中形成包膜的HCV病毒粒子。少