State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
J Virol. 2014 Mar;88(6):3379-91. doi: 10.1128/JVI.02782-13. Epub 2014 Jan 3.
Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex.
The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans-complementation enhances our understanding of NS4B in flavivirus replication.
黄病毒的复制是由一个复杂的机制介导的，该机制由病毒酶、非酶病毒蛋白和宿主因子组成。许多非酶病毒蛋白，如 NS4B，与内质网膜相关。这些膜蛋白如何在病毒复制中发挥作用还知之甚少。在这里，我们报告了一种表达和纯化登革热病毒（DENV）和西尼罗河病毒 NS4B 蛋白的稳健方法。NS4B 蛋白在大肠杆菌中表达，在十二烷基麦芽糖苷（DDM）去污剂胶束中重组，并纯化至>95%的纯度。凝胶过滤、化学交联和多角度光散射实验证明，重组 NS4B 蛋白在体外二聚化。当蛋白单独在细胞中表达以及在感染 DENV 型 2（DENV-2）的细胞中表达时，也检测到 NS4B 的二聚体形式。突变分析表明，细胞质环（氨基酸 129 至 165）和 C 末端区域（氨基酸 166 至 248）负责 NS4B 二聚化。转互补实验表明：（i）两个包含不同 NS4B 致死突变的基因组长度 RNA 不能相互转互补；（ii）NS4B 突变 RNA 的复制缺陷可以在含有 DENV-2 复制子的细胞中恢复；（iii）单独表达野生型 NS4B 蛋白不足以恢复 NS4B 突变 RNA 的复制。总之，结果表明，致死性 NS4B 突变 RNA 的转互补需要来自复制复合物的野生型 NS4B 呈现。
所报道的表达和纯化系统使得研究黄病毒 NS4B 蛋白的生物化学和结构成为可能。黄病毒 NS4B 二聚化的发现和 NS4B 二聚化重要区域的定位为通过阻断 NS4B 二聚化来抑制病毒复制提供了可能性。在复制复合物背景下 NS4B 对成功转互补的要求增强了我们对 NS4B 在黄病毒复制中的理解。
