Formation of Vesicular Stomatitis Virus Pseudotypes Bearing Surface Proteins of Hepatitis B Virus
Formation of Vesicular Stomatitis Virus Pseudotypes Bearing Surface Proteins of Hepatitis B Virus
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携带乙型肝炎病毒表面蛋白的水泡性口炎病毒假型的形成
DOI：
10.1128/jvi.79.19.12566-12574.2005
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发表时间：
2005
影响因子：
5.4
通讯作者：
H. Hoshino
中科院分区：
文献类型：
--
作者：
M. Saha;A. Tanaka;Atsushi Jinno;N. Shimizu;K. Tamura;M. Shinagawa;J. Chiba;H. Hoshino
摘要组织培养中乙型肝炎病毒（B）的增殖和滴定一直是一个难题。我们检查是否水泡性口炎病毒（VSV）的假型携带HBV表面（HBs）蛋白感染人类细胞系可以制备。为此，制备了HBV的三种表面蛋白L、M和S的表达质粒。然后用这些质粒单独或以不同组合转染293 T细胞。用表达绿色荧光蛋白（GFP）的重组VSVΔG*-G感染表达HBs蛋白的293 T细胞，以制备VSV假型。收获培养物上清液和细胞并短时间超声处理。通过定量用抗VSV血清和抗HBs蛋白的小鼠单克隆抗体（MAb）中和后表达GFP的细胞的数量来确定新鲜收获的上清液的感染性。在检测对HBV假型样品的敏感性的14种细胞系中，HepG 2、JHH-7和293 T细胞被判定为最敏感。即，在不存在和存在抗-HBs S MAbs的情况下用抗-VSV中和并在HepG 2细胞上滴定的培养上清液样品的感染单位（IU）分别为1，000至4，000 IU/ml和200至400 IU/ml，表明存在VSVΔG*（HBV）假型。通过用乳铁蛋白或硫酸葡聚糖处理来抑制这种感染性。用胰蛋白酶或衣霉素预处理细胞抑制假型样品的铺板。HBV假型可用于分析HBV感染的早期步骤，包括HBV的进入机制。
ABSTRACT It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVΔG*-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVΔG*(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.
DOI：
10.1073/pnas.94.26.14764
发表时间：
1997-12-23
影响因子：
11.1
作者：
Takada, A;Robison, C;Kawaoka, Y
通讯作者：
Kawaoka, Y