克氏原螯虾多配体蛋白聚糖syndecan作为膜受体蛋白介导WSSV侵染的分子机制研究

White spot syndrome virus (WSSV) has brought great harm to the crayfish breeding industry, studying the infection mechanism of WSSV will help prevention and treatment of diseases. In previous study, the applicant found that syndecan could bind to WSSV and was associated with proliferation and replication of WSSV. By inhibiting the activity of heparan sulfate chain HS of syndecan, the copy number of WSSV can be significantly reduced. Based on these data, we hypothesized that the syndecan protein may act as a membrane receptor protein and play a key role in the process of mediating WSSV infection by HS sugar chains. Therefore, we use the Procambarus clarkii as the research object, and analyze the relationship between the inhibition expression and overexpression of syndecan and WSSV proliferation and replication. We also detect the interaction between syndecan and WSSV proteins. These results will prove that syndecan is a membrane receptor protein of WSSV. We also uas the inhibition and enhancement of HS activity and study of the dynamic process of HS entry into WSSV to analyze the relationship between changes in HS sugar chains and block WSSV infection. The results will systematically elucidate the molecular mechanism of syndecan protein on the cell membrane as a receptor-mediated WSSV invasion, and provide new ideas for finding anti-WSSV drug targets and developing antiviral drugs.

白斑综合征病毒(WSSV)给小龙虾养殖产业带来了巨大的危害，研究WSSV的侵染机制有助于病害的防治。申请人前期研究中发现syndecan能够与WSSV结合，并与WSSV的增殖复制相关，通过抑制syndecan的硫酸乙酰肝素糖链HS的活性能够显著降低WSSV的拷贝数。据此我们提出假说：syndecan蛋白可能作为膜受体蛋白，通过HS糖链在介导WSSV侵染细胞的过程中发挥着关键性的作用。因此本项目以克氏原螯虾为研究对象，通过分析抑制表达和过表达syndecan与WSSV增殖复制的关系以及蛋白间的互作关系，来证实syndecan是WSSV侵染的膜受体蛋白；并通过抑制与增强HS活性以及研究HS在WSSV入胞的动态过程，来分析HS糖链的变化与阻滞WSSV感染的关系。研究结果将系统阐明细胞膜上syndecan蛋白作为受体介导WSSV入侵的分子机制，为寻找抗WSSV药物靶点、开发抗病毒药物提供新思路。

研究团队前期发现克氏原螯虾中的syndecan蛋白可能参与白斑综合征病毒WSSV的侵染过程，但WSSV是如何通过syndecan蛋白来感染宿主的侵染机制尚不明确。Syndecan蛋白是一种细胞膜蛋白，其可能作为膜受体蛋白，通过硫酸乙酰肝素HS糖链在介导WSSV侵染细胞过程中发挥着关键性的作用。因而本项目首先研究克氏原螯虾syndecan蛋白表达特性及与WSSV增殖复制的关系，然后研究了syndecan蛋白的HS糖链活性与阻滞WSSV侵染的关系，最后研究了syndecan蛋白与WSSV病毒间的互作关系。研究结果表明抑制syndecan的表达和活性能够显著降低WSSV的感染效率，而增强syndecan的表达和活性则能够提高WSSV的感染效率，结果证实了syndecan蛋白是有助于WSSV的感染。利用HS糖链富含负电荷的特性，分别通过多聚赖氨酸和肝素酶处理，则能够显著降低螯虾体内的WSSV拷贝数；而外源性注射HS糖链，则会显著增加螯虾体内WSSV的拷贝数，结果表明syndecan通过连接在其表面的HS糖链来实现其作为膜受体或共受体促进WSSV的感染。为了进一步探究syndecan与WSSV的互作关系，通过Far-western，Pull-down，ELISA和LCMS/MS等方法，证实了syndecan蛋白能够与WSSV病毒颗粒在体外条件下结合互作，通过质谱分析这些发生结合作用的蛋白可能是WSSV的“wsv329”，“wsv271”，“ORF37”和“WSV012”蛋白。另外研究指出syndecan蛋白能够通过胞内的信号传导介导核转录因子Akirin基因的激活，从而调控螯虾体内抗菌肽相关基因ALF、crustin、Proph、Lectin、thyβ4等基因的表达。同时syndecan能够同一种分泌的胰蛋白酶样丝氨酸蛋白酶Masquerade (Mas)基因有互作关系，而Mas能够通过与细菌和WSSV结合并激活酚氧化酶系统来提高机体的免疫反应，从而清除体内病原。因此本研究明确了syndecan蛋白可作为WSSV侵染细胞的关键膜受体蛋白发挥作用，并通过对其蛋白的HS糖链活性抑制能够达到阻滞WSSV感染的目的。研究结果揭示了鳌虾通过syndecan蛋白与WSSV侵染和免疫防御的互作关系，不仅丰富了WSSV侵染的分子机制，而且还为寻找抗WSSV药物靶点、开发抗病药物提供了新思路。