波形蛋白与Ⅱ型登革病毒非结构蛋白的相互作用及其在病毒复制中功能的研究

Dengue virus is one of the major emerging arboviral pathogen that cause serious epidemics around the world. Currently, there is no effective drugs for treatment. The viral replication complex (RC), where the DENV RNA replicates, is formed around ER membrane after DENV entry. RC formation is the key step for viral replication, and is mainly composed of viral proteins, viral RNA and host proteins. At present, the formation of RC remains to be elucidated, especially the mechanism of recruitment for the DENV non-structural protein to RC. Cytoskeleton plays an important role in intracellular transport, maintenance of morphology, support and anchoring organelles. It is reported that the cytoskeleton vimentin interacts with DENV nonstructural protein NS1 and NS4A and recruits the NS4A to RC. Our previous studies also show that vimentin interacts with NS3. Here, we proposed that vimentin may serve as the scaffold and play a key role in the recruitment of viral nonstructural proteins during the formation of DENV RC. We intend to determine the interaction between vimentin and DENV nonstructural proteins, and the role in the formation of viral RC, and to further screen for the small peptides that inhibit this interaction. This project would clarify the role of vimentin in RC formation and non-structural proteins recruitment, help to elucidate the replication mechanism of flaviviruses represented by DENV, and provide new ideas and new targets for anti-DENV drug design.

登革病毒是引发全球范围内严重疫情的虫媒病毒之一，尚无有效的治疗药物。内质网膜内陷形成复制复合物（RC）是DENV复制的关键环节，主要由病毒蛋白、病毒RNA和宿主蛋白构成。目前，DENV非结构蛋白从翻译位点向RC募集的机制尚不清楚。细胞骨架在维持细胞形态、囊泡运输、支撑和锚定细胞器等方面有重要作用。研究发现，细胞骨架蛋白-波形蛋白与DENV NS1和NS4A相互作用，并且作为桥梁介导NS4A向RC募集。我们前期研究表明，波形蛋白与NS3也有相互作用。据此推测，波形蛋白在RC形成过程中起到“脚手架”的作用并参与募集病毒非结构蛋白。本课题组拟明确波形蛋白与非结构蛋白的相互作用位点，及其在病毒RC形成过程中的作用，并筛选出抑制这种相互作用而影响病毒复制的小肽抑制剂。本课题的完成可明确波形蛋白与非结构蛋白相互作用如何参与RC的形成过程，完善DENV的复制机制，为抗DENV药物设计提供新思路和新靶点。

登革病毒（DV）是引发全球范围内严重疫情的虫媒病毒之一，尚无有效的治疗药物。内质网膜内陷形成的复制复合物（RC）是DV复制的关键环节，细胞骨架波形蛋白（vimentin）可能在RC的形成过程中可能起到重要作用。本课题的主要研究内容包括明确vimentin与非结构蛋白的相互作用位点，及其在病毒RC形成过程中的作用，并筛选出抑制这种相互作用而影响病毒复制的小分子抑制剂。我们的研究结果表明DV dsRNA和非结构蛋白 NS1, NS2b, NS3, NS4a, NS4b均与vimentin有相互作用关系，DV感染可引起vimentin在细胞内重排；此外，vimentin是AKT激酶的下游底物，小分子抑制剂AT13148通过抑制AKT激酶活性抑制了vimentin磷酸化；AT13148在DV入胞后发挥抗病毒作用，IC50为569 nM，具有良好的成药性。 总的来讲，本课题明确了宿主蛋白vimentin与DV非结构蛋白的相互作用关系，及其参与DV复制的分子机制，并筛选出靶向vimentin的抗病毒小分子药物且阐明了该分子抗病毒感染的机制，为抗DV药物设计提供了新思路和新靶点。