Chen Hongying, Wurm Torsten, Britton Paul, Brooks Gavin, Hiscox Julian A
Virology Group, School of Animal and Microbial Sciences, The University of Reading, United Kingdom.
J Virol. 2002 May;76(10):5233-50. doi: 10.1128/jvi.76.10.5233-5250.2002.
Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.
在病毒感染的原代细胞以及转染了表达核蛋白（N蛋白）质粒的细胞中，冠状病毒核蛋白（N蛋白）定位于细胞质和核仁（一种亚核结构）。核仁是核糖体生物合成的场所，并且隔离细胞周期调节复合物。核仁的两个主要成分是纤维蛋白原和核仁素。这些蛋白质参与核仁组装和核糖体生物合成，并作为蛋白质导入核仁的分子伴侣。我们发现，在感染禽冠状病毒传染性支气管炎病毒（IBV）的原代细胞以及表达IBV或小鼠肝炎病毒N蛋白的连续细胞系中，纤维蛋白原会发生重排。N蛋白和纤维蛋白原-绿色荧光蛋白融合蛋白都共定位于核周区域和核仁。下拉实验表明，IBV N蛋白与核仁素相互作用，因此为冠状病毒N蛋白如何定位于核仁提供了一种可能的解释。核仁以及定位于核仁的蛋白质与细胞生长-细胞周期调节有关。将表达IBV N蛋白的细胞与对照细胞进行比较表明，表达N蛋白的细胞生长延迟。该结果不能归因于细胞凋亡。对这些细胞的形态学分析表明，胞质分裂受到破坏，这一观察结果随后在感染IBV的原代细胞中也发现了。因此，冠状病毒可能会在间期延迟细胞周期，而在间期病毒mRNA可以进行最大程度的翻译。
