Structural and Biochemical Characterisation of Bunyavirus L proteins.

布尼亚病毒属于具有负方向的分段RNA基因组的病毒组。许多新出现的病毒病原体属于布尼亚病毒科。它们能够感染人类和动物，并能在宿主中引起严重疾病。由于最近的爆发，从新闻中得知的突出例子是汉坦病毒、裂谷热病毒、施马伦贝格病毒和克里米亚-刚果出血热病毒。 这些病毒生命周期中的一个关键组成部分是病毒L蛋白，一种含有病毒RNA依赖性RNA聚合酶的大型多结构域蛋白。由于其大尺寸和复杂的结构，关于L蛋白的数据很少。深入了解这台大型分子机器将为医学研究提供重要基础。因此，我们的目标是研究纯化的L蛋白使用跨学科的方法结合经典生物化学与病毒学和结构生物学。具体目标包括（1）在细菌细胞中重组表达和纯化布尼亚病毒L蛋白片段，（2）对这些蛋白进行生物化学和结构研究，以了解其酶功能并确定潜在的结构同源物。另外（3）我们将利用昆虫细胞或哺乳动物细胞表达系统纯化全长L蛋白用于功能分析。最后（4）我们将使用基于细胞的复制子测定来分析L蛋白的特定残基或区域在转录和复制中的作用。这些目标之一的结果将有助于优化其他目标的实验设计。总之，所有的结果放在一起将提供一个完整的观点的作用，L蛋白在病毒的生命周期。

Bunyaviruses belong to the group of viruses that possess a segmented RNA genome in negative orientation. Many of the newly emerged viral pathogens belong to the family of Bunyaviridae. They are capable of infecting both humans and animals and can cause severe diseases in their hosts. Prominent examples known from the news due to recent outbreaks are Hantaviruses, the Rift-Valley Fever Virus, the Schmallenberg Virus and the Crimean-Congo-Hemorrhagic Fever Virus. A key component in the lifecycle of these viruses is the viral L protein, a large multi-domain protein containing the RNA-dependant RNA polymerase of the virus. Due to its large size and complex structure, data on the L protein is scarce. A thorough understanding of this large molecular machine will provide an important basis for medical research. We therefore aim at studying the purified L protein using a interdisciplinary approach combining classical biochemistry with virology and structural biology. Specific objectives include (1) the recombinant expression and purification of fragments of the Bunyavirus L protein in bacterial cells, (2) the biochemical and structural investigation of these proteins to understand their enzymatic function and determine potential structural homologs. In addition (3) we will purify full-length L protein using insect cell or mammalian cell expression systems for functional analyses. Finally (4) we will use cell based replicon assays to analyse the role of specific residues or regions of the L protein in transcription and replication. Results from one of these objectives will aid in optimization of the experimental design for the other objectives. In summary, all results taken together will provide an integrated view of the role of the L protein in the viral life cycle.