Structural and Biochemical Characterisation of Bunyavirus L proteins.

布尼亚病毒属于具有负向分段RNA基因组的病毒群。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，并可在其宿主中引起严重疾病。由于最近爆发的疫情而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成分是病毒L蛋白，这是一种含有病毒依赖RNA的RNA聚合酶的大型多结构域蛋白。由于L蛋白的体积大，结构复杂，有关其的资料很少。深入了解这种大分子机器将为医学研究提供重要的基础。因此，我们的目标是利用跨学科的方法，结合经典生物化学与病毒学和结构生物学来研究纯化的L蛋白。具体目的包括：(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)对这些蛋白进行生化和结构研究，以了解它们的酶功能并确定潜在的结构同源物。此外(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蛋白进行功能分析。最后(4)我们将使用基于细胞的复制子分析来分析L蛋白的特定残基或区域在转录和复制中的作用。其中一个目标的结果将有助于优化其他目标的实验设计。总之，所有的结果综合起来将提供一个综合的观点，在病毒生命周期中的作用的L蛋白。

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.