Kumar Purnima, Gunalan Vithiagaran, Liu Boping, Chow Vincent T K, Druce Julian, Birch Chris, Catton Mike, Fielding Burtram C, Tan Yee-Joo, Lal Sunil K
Virology Group, International Centre for Genetic Engineering and Biotechnology, P. O. Box: 10504, Aruna Asaf Ali Road, New Delhi 110067, India.
Virology. 2007 Sep 30;366(2):293-303. doi: 10.1016/j.virol.2007.04.029. Epub 2007 May 25.
Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.
严重急性呼吸综合征（SARS）冠状病毒（SARS-CoV）在2003年于世界多个地区引发了严重疫情。据预测，SARS-CoV基因组包含14个功能性开放阅读框（ORF）。第一个ORF（1a和1b）编码一种大型多聚蛋白，该蛋白可被切割成非结构蛋白（nsp）。其他ORF编码四种结构蛋白（刺突蛋白、膜蛋白、核衣壳蛋白和包膜蛋白）以及八种SARS-CoV特异性辅助蛋白（3a、3b、6、7a、7b、8a、8b和9b）。在本报告中，我们克隆了SARS-CoV预测的nsp8基因和ORF6基因，并研究了它们相互作用的能力。我们在酵母双杂交系统中将这两种蛋白表达为融合蛋白以证明蛋白-蛋白相互作用，并通过酵母遗传杂交进行了测试。此外，通过在2-氨基三唑浓度递增梯度上挑战阳性相互作用克隆的生长来测量相互作用的强度。然后通过在体外偶联转录翻译系统中分别表达这两种蛋白以及在哺乳动物细胞中进行共免疫沉淀来验证这种相互作用。最后，在感染SARS-CoV的Vero E6哺乳动物细胞中进行共定位实验以确认nsp8-ORF6相互作用。据我们所知，这是关于SARS-CoV辅助蛋白与nsp8之间相互作用的首次报道，我们的研究结果表明ORF6蛋白可能在病毒复制中发挥作用。
