Ortego Javier, Ceriani Juan E, Patiño Cristina, Plana Juan, Enjuanes Luis
Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain.
Virology. 2007 Nov 25;368(2):296-308. doi: 10.1016/j.virol.2007.05.032. Epub 2007 Aug 10.
A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-DeltaE) has been engineered. This deletion mutant only grows in cells expressing E protein (E(+) cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-DeltaE infected BHK-pAPN-E(-) cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E(-) cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-DeltaE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-DeltaE subcellular localization by confocal and immunoelectron microscopy in infected E(-) cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation.
一种缺失E基因的重组传染性胃肠炎冠状病毒（rTGEV-DeltaE）已被构建。这种缺失突变体仅在表达E蛋白的细胞（E(+)细胞）中生长，这表明E是TGEV复制的必需基因。对感染rTGEV-DeltaE的BHK-pAPN-E(-)细胞进行电子显微镜研究表明，仅组装了未成熟的细胞内病毒粒子。这些病毒粒子没有感染性，并且在BHK-pAPN-E(-)细胞中不会分泌到细胞外培养基中。通过核糖核酸酶-金和免疫电子显微镜进行的RNA和蛋白质组成分析表明，rTGEV-DeltaE病毒粒子含有RNA，并且除了缺失的E蛋白外，还含有所有TGEV结构蛋白。然而，病毒粒子的完全成熟被阻断。通过共聚焦和免疫电子显微镜对感染的E(-)细胞中rTGEV-DeltaE的亚细胞定位研究表明，在没有E蛋白的情况下，病毒运输在中间区室中停滞。因此，TGEV中E蛋白的缺失导致了两种作用，一是阻断了病毒在分泌途径膜中的运输，二是阻止了病毒的完全成熟。
