Cologna R, Spagnolo J F, Hogue B G
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Virology. 2000 Nov 25;277(2):235-49. doi: 10.1006/viro.2000.0611.
The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.
冠状病毒核衣壳（N）蛋白是病毒粒子的主要结构成分，它与基因组RNA结合形成螺旋核衣壳。N似乎是一种多功能蛋白，因为数据还表明该蛋白可能参与病毒RNA的复制和翻译。所有这些功能可能都涉及N与病毒RNA之间的相互作用。作为理解N如何与病毒RNA相互作用的第一步，我们在牛冠状病毒（BCV）和小鼠肝炎病毒（MHV）缺陷基因组中绘制了高效N结合位点。体内和体外试验均用于研究BCV和MHV N蛋白与病毒和非病毒RNA的结合。在牛冠状病毒（BCV）感染的细胞和瞬时表达N蛋白的细胞中检测到N-病毒RNA复合物。滤膜结合用于绘制Drep内的N结合位点，Drep是一种BCV缺陷基因组，在辅助病毒存在下进行复制和包装。在Drep内N ORF 3'端的核苷酸1441和1875之间鉴定出一个高效N结合位点。为了进行比较，还绘制了小鼠肝炎冠状病毒（MHV）缺陷干扰（DI）RNA MIDI-C的N结合位点。对于包含MHV包装信号（nts 3949-4524）的区域以及MIDI-C内MHV N ORF 3'端的一个区域（nts 4837-5197）的RNA转录本，测量到的结合效率与Drep相似。与全长MIDI-C转录本（约5500 nts）以及来自MIDI-C基因1a区域（nts 935-1986）且排除包装信号的约1 kb转录本的结合均显著高于较小转录本的结合效率。这是首次鉴定出BCV的N结合序列。这也是首次报道证明N在体外与MHV基因组中除包装信号和前导序列之外的序列相互作用。数据清楚地表明，N与冠状病毒RNA的结合比与非病毒RNA的结合更有效。这些结果对N的多功能作用具有重要意义。
