Geigenmüller-Gnirke U, Nitschko H, Schlesinger S
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
J Virol. 1993 Mar;67(3):1620-6. doi: 10.1128/JVI.67.3.1620-1626.1993.
The capsid protein of Sindbis virus has multiple functions in the life cycle of the virus. One essential function is to interact with the genomic RNA of the virus to form the nucleocapsid. The experiments described in this article define a region of the protein that is required for binding to Sindbis virus RNA. The assay we used measured the binding of in vitro-translated proteins to RNA on the basis of their migration with the RNA during electrophoresis in an agarose gel. Binding to RNA showed specificity; more protein bound to an RNA containing the previously defined packaging signal in Sindbis virus RNAs than to a similar RNA lacking this sequence. We were able to produce a variety of deleted forms of the capsid protein by constructing cDNAs with in-frame deletions throughout the coding region of the capsid protein gene. These cDNAs were then transcribed into mRNAs and translated in vitro. C-terminal deletions in the capsid protein were obtained by preparing transcripts from cDNAs linearized at sites within the coding region. Our studies identified a 32-amino-acid region that is essential for the specificity in RNA binding, and they defined a 68-amino-acid minimal sequence which displays almost the complete specific RNA binding activity of the intact Sindbis virus capsid protein containing 264 amino acids.
辛德毕斯病毒的衣壳蛋白在病毒生命周期中具有多种功能。其中一个重要功能是与病毒的基因组RNA相互作用形成核衣壳。本文所述的实验确定了该蛋白中与辛德毕斯病毒RNA结合所需的一个区域。我们使用的检测方法是基于体外翻译的蛋白质在琼脂糖凝胶电泳过程中与RNA一起迁移的情况来测量其与RNA的结合。与RNA的结合表现出特异性；与含有辛德毕斯病毒RNA中先前定义的包装信号的RNA结合的蛋白质比与缺乏该序列的类似RNA结合的蛋白质更多。通过构建在衣壳蛋白基因编码区带有框内缺失的cDNA，我们能够产生多种衣壳蛋白的缺失形式。然后将这些cDNA转录成mRNA并进行体外翻译。通过从编码区内位点线性化的cDNA制备转录本，获得了衣壳蛋白的C端缺失。我们的研究确定了一个对RNA结合特异性至关重要的32个氨基酸的区域，并定义了一个68个氨基酸的最小序列，该序列显示出几乎与含有264个氨基酸的完整辛德毕斯病毒衣壳蛋白完全相同的特异性RNA结合活性。
