Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging
Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging
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DOI：
10.1128/jvi.01957-13
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发表时间：
2014-01-01
影响因子：
5.4
通讯作者：
Tautz, Norbert
中科院分区：
文献类型：
--
作者：
Isken, Olaf;Langerwisch, Ulrike;Tautz, Norbert
牛病毒性腹泻病毒(BVDV)非结构蛋白5A(NS5A)是一种具有RNA结合活性的亲水性磷蛋白，是病毒复制酶的重要组成部分。计算机分析表明，NS5A包含由两个低复杂性序列(LCS)相互连接的三个结构域。虽然结构域I含有两个功能决定因素，一个是对膜结合重要的N端两亲性螺旋，另一个是RNA复制所必需的锌结合位点，但NS5A的C末端半部分的结构和功能仍不明确。在这项研究中，我们介绍了一组覆盖NS5A C-末端一半的10个氨基酸缺失。在高效单顺反子复制子的背景下，在RNA复制方面，LCS I和结构域II的N-末端部分以及结构域III中的缺失是可以容忍的。当被引入双顺反子复制子时，只允许LCS I和结构域II的N-末端部分的缺失。在病毒全长基因组的背景下，这些突变允许残留病毒粒子的形态发生。基于这些数据，构建了编码NS5A变异体的功能性单顺反子BVDV复制子，并插入了荧光蛋白mCherry。活细胞成像显示，NS5A-mCherry的一部分定位于脂滴表面。综上所述，这项研究为BVDV NS5A的功能提供了新的见解。此外，我们还建立了第一个表达荧光NS5A-mCherry的瘟疫病毒复制子，通过活细胞成像直接显示了具有功能的病毒复制复合体。
Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.