Herod Morgan R, Salim Omar, Skilton Rachel J, Prince Cynthia A, Ward Vernon K, Lambden Paul R, Clarke Ian N
Molecular Microbiology Group, University of Southampton, Southampton, United Kingdom.
Otago School of Medical Sciences, Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
PLoS One. 2014 Mar 5;9(3):e90679. doi: 10.1371/journal.pone.0090679. eCollection 2014.
Investigations into human norovirus infection, replication and pathogenesis, as well as the development of potential antiviral agents, have been restricted by the lack of a cell culture system for human norovirus. To date, the optimal cell culture surrogate virus model for studying human norovirus biology is the murine norovirus (MNV). In this report we generate a tetracycline-regulated, inducible eukaryotic cell system expressing the entire MNV ORF1 polyprotein. Once induced, the MNV ORF1 polyprotein was faithfully processed to the six mature non-structural proteins that predominately located to a discrete perinuclear region, as has been observed in active MNV infection. Furthermore, we found that expression of the ORF1 polyprotein alone was sufficient to induce apoptosis, characterised by caspase-9 activation and survivin down-regulation. This cell line provides a valuable new tool for studying MNV ORF1 non-structural protein function, screening for potential antiviral agents and acts as a proof-of-principle for such systems to be developed for human noroviruses.
由于缺乏用于人类诺如病毒的细胞培养系统，对人类诺如病毒感染、复制和发病机制的研究以及潜在抗病毒药物的开发一直受到限制。迄今为止，用于研究人类诺如病毒生物学的最佳细胞培养替代病毒模型是鼠诺如病毒（MNV）。在本报告中，我们构建了一个四环素调控的、可诱导的真核细胞系统，该系统表达完整的MNV ORF1多聚蛋白。一旦诱导，MNV ORF1多聚蛋白会被准确加工成六种成熟的非结构蛋白，这些蛋白主要定位于离散的核周区域，这与在活跃的MNV感染中观察到的情况一致。此外，我们发现单独表达ORF1多聚蛋白就足以诱导细胞凋亡，其特征为半胱天冬酶-9激活和生存素下调。该细胞系为研究MNV ORF1非结构蛋白功能、筛选潜在抗病毒药物提供了一个有价值的新工具，并为开发用于人类诺如病毒的此类系统提供了原理验证。
