Functional characterization of cellular RNA-binding proteins that support Flavivirus replication

黄病毒复制的必要前提是病毒正链RNA基因组的环化，这使得病毒编码的复制酶能够启动RNA合成。在早期的研究中，我们将富au元素RNA结合蛋白1 （AUF1）的p45亚型定义为一种细胞宿主因子，该因子在很大程度上支持蚊子传播的西尼罗病毒（WNV）在人细胞中的RNA复制过程。一组互补的实验数据表明，AUF1 p45具有RNA伴侣活性，通过破坏病毒RNA 3'端的茎结构，支持RNA环化和病毒RNA合成。此外，该蛋白被证明具有RNA退火活性，这可能有助于病毒基因组5‘和3’区互补环化序列的杂交。这项应用的研究旨在进一步详细了解AUF1 p45的活性，以及该蛋白在作为宿主因子作用于黄病毒rna时的潜在蚊子类似物的活性。将解决三个相互关联的问题：(1)AUF1 p45蛋白一侧和黄病毒RNA一侧的分子决定因素是什么，这些决定因素使蛋白质一方面破坏病毒RNA的稳定性，另一方面支持RNA退火？(2) AUF1 p45是否与其他黄病毒（如西尼罗河病毒相关的登革热病毒（DENV））有类似的作用？(3)蚊子细胞是否编码与人类AUF1 p45具有同源功能的类似宿主因子？拟议的项目预计将在两个方面产生新的知识。一方面，我们将进一步了解细胞rna结合蛋白（RBP）如何调节病毒复制机制的分子机制。此外，我们期望获得关于rbp / RNA伴侣如何控制RNA分子结构和功能的一般机制的重要信息。

An essential prerequisite for the replication of Flaviviruses is the cyclization of the viral positive-strand RNA genome, which enables the virus-encoded replicase to initiate RNA synthesis. During earlier studies, we defined the p45 isoform of the AU-rich element RNA-binding protein 1 (AUF1) as a cellular host factor that considerably supports the RNA replication process of the mosquito borne Flavivirus West Nile virus (WNV) in human cells. A set of complementary experimental data indicated that AUF1 p45 has an RNA chaperone activity, which supports RNA cyclization and viral RNA synthesis by destabilizing a stem-structure at the viral RNA's 3'end. Moreover, the protein was shown having an RNA annealing activity, which is suspected to assist the hybridization of complementary cyclization sequences in the 5'- and 3'regions of the viral genome. The proposed studies of this application aim at gaining a further detailed picture on the activities of AUF1 p45 and of potential mosquito analogues of the protein while operating as host factors on flaviviral RNAs. Three interrelated questions will be addressed: (1) What are the molecular determinants on the side of the AUF1 p45 protein and on the side of the flaviviral RNA that enable the protein to destabilize the viral RNA on the one and to support RNA annealing on the other hand? (2) Does AUF1 p45 act in a similar way with other flaviviruses, e.g. the WNV-related Dengue virus (DENV)? (3) Do mosquito cells encode similar types of host factors that have homologous functions as the human AUF1 p45? The proposed project is expected to generate new knowledge in two respects. On the one hand, we will obtain additional insights into the molecular mechanisms on how a cellular RNA-binding protein (RBP) is capable to modulate the activity of viral replication machineries. Moreover, we anticipate gaining important information on the general mechanisms of how RBPs / RNA chaperones control the structures and functions of RNA molecules.