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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Replication and packaging of Norwalk virus RNA in cultured mammalian cells
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Replication and packaging of Norwalk virus RNA in cultured mammalian cells

机译：在哺乳动物细胞中复制和包装诺沃克病毒RNA

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Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/ T7). Expressed genomic RNA was found to replicate; NV sub-genomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCI) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.
机译：人诺如病毒是非细菌性肠胃炎的最常见原因，其特征是感染率高，感染剂量低以及在宿主外部的异常稳定性高。然而，人类诺如病毒的研究由于缺乏细胞培养系统和小型动物感染模型而受到阻碍。诺沃克病毒（NV）是人类诺如病毒的原型毒株。我们在这里报告了NV病毒RNA的复制及其包装，通过胞内表达的NV病毒RNA的天然形式的胞内表达，通过使用复制缺陷型痘苗病毒MVA编码噬菌体，而没有表达载体衍生的外来核苷酸序列T7 RNA聚合酶（MVA / T7）。表达的基因组RNA被发现可以复制。通过使用从基因组RNA表达的NV非结构蛋白，从基因组RNA转录NV亚基因组RNA，随后将其翻译为NV衣壳蛋白VP1。将病毒基因组RNA包装到哺乳动物细胞中产生的病毒颗粒中。含有基因组RNA的病毒颗粒的氯化铯（CsCI）密度梯度曲线与从粪便中纯化的NV相似。这些观察结果表明，这里构建的NV cDNA是生物学感染性克隆，并且哺乳动物细胞具有复制NV基因组RNA的能力。这项工作建立了一个基于哺乳动物细胞的系统，用于分析人类诺如病毒的复制，因此使研究哺乳动物细胞中的抗病毒剂成为可能。

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