登革病毒E蛋白与整合素β3相互作用研究

Dengue virus (DV) which causes classical dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is one of the most important arthropodborne viruses infecting humans. The pathogenesis of DV infection is not completely clear and currently there is no vaccine or specific antivirals available for humans. Integrins are glycoproteins widely distributed in the surface of cell membranes. Previous studies have showed that integrin β3 may serve as receptor or coreceptor mediated the entry of DV into host cell. DV envelope (E) protein, the largest structural glycoprotein, is responsible for host cell attachment, entry, and virus mediated cell membrane fusion. It may be the important structural protein interacting with host integrin β3. Our Previous study showed that DV E protein and integrin β3 bound to each other with a very high affinity. However, the mechanism of this interaction remains unknown. To elucidate the interaction between integrin β3 and E protein, the experiments are designed as follows: 1. Determine the interaction between integrin β3 and E protein by GST pull-down, co-immunoprecipitation and surface plasmon resonance (SPR); 2. Determine the binding sites of DV E protein to integrin β3 by SPR and PCR site-directed mutagenesis; 3. Use synthetic peptide to block DV infection. This study aims to provide evidences to well understand the pathogenesis and to screen the drugs of DV infection.

由登革病毒（Dengue virus，DV）感染引起的登革热和登革出血热是当前流行最为广泛的虫媒病毒病，其发病机制不明，也无特异性疫苗和药物用于防治。整合素是广泛分布于细胞表面的糖蛋白，前期我们已证明整合素β3可能作为病毒受体或共受体参与DV进入靶细胞的过程，而作为吸附蛋白的E蛋白可能是DV与整合素β3相互作用的重要组分，预实验发现DV E蛋白与整合素β3有很强的亲和力，但其具体作用方式和机制不清。为此, 本课题拟从以下几方面进行研究：1.利用GST pull-down、免疫共沉淀和表面等离子共振surface plasmon resonance (SPR)法，明确整合素β3与病毒E蛋白相互作用的方式；2.利用SPR和点突变法，确定E蛋白与整合素β3作用的具体位点；3.利用合成肽阻断实验，明确作用位点在病毒感染过程中的意义。期望本研究能够为阐明DV感染机制，进而设计抗DV药物提供依据。

由登革病毒（Dengue virus，DV）感染引起的登革热和登革出血热是当前流行最为广泛的虫媒病毒病，其发病机制不明，也无特异性疫苗和药物用于防治。目前，参与DV感染的受体或关键分子尚不清楚，我们推测DV可能通过与E蛋白某些区域或位点与整合素β3相互作用而感染细胞。本研究发现DV2感染可以提高整合素β3在HUVEC中的表达，利用激光共聚焦显微镜可观察到整合素β3和DV E蛋白在细胞内的共定位；利用酵母双杂法和表面等离子共振surface plasmon resonance (SPR)法对整合素β3与E蛋白相互作用研究发现：整合素β3与DV2 E蛋白的确存在相互作用。对DV2 E蛋白分段表达，并通过SPR法研究其与整合素β3的亲和力，发现DV2E蛋白的Ⅲ区段（E3）与整合素β3有较强亲和力。对E3区段合成小分子肽，并研究其对DV2感染的抑制作用，发现P4(C333-V347)和P7 (G381-K394)与亲和力较强，且对DV2感染有明显抑制作用，其抑制效率可达60%以上，且有明显的剂量依赖性。本研究证实：DV2通过E3区段与整合素β3发生相互作用，其作用位点在C333-V347和 G381-K394 区，且阻断该位点可抑制DV2的感染。本研究为阐明登革热的发生机制提供了重要的实验室资料，并有望为研究抗病毒药物的研究提供新的靶点。