SVCV入侵机制及动力学特征研究

Spring viraemia of carp virus (SVCV) is the causative pathogen of the outbreaks of haemorrhagic disease responsible for the significant mortality in carps. As an RNA virus, SVCV shares a high level of genomic plasticity, and currently, there are no effective vaccines or drugs for this virus. SVCV seriously threaten the aquaculture industry, causing great economic losses in China. However, the cell entry mechanism of SVCV remains uncharacterized. By using endocytic inhibitors and anti-M antibody, our primary studies indicated that SVCV entered CO cells via the clathrin-mediated endocytic pathway and macropinocytosis but not via caveola-dependent endocytosis, and the cell entry process was depent on actin microfilaments and microtubules. With quantum dots labeled SVCV, we also observed that the entry process took only about 5 min. In this proposed project, firstly, we plan to clarify the endocytic pathway of SVCV by using added endocytic specific inhibitors and other perturbants, siRNA and mutant counstructs of endocytic-related crucial proteins. Meanwhile, we will investigate the interactions between SVCV and the host cell cytoskeleton networks. Then, we will visulize the dynamic endocytic process of SVCV by quantum dots labeled virus live-cell imaging. Next, we will verify the screened inhibitors, siRNAs and protein mutants of SVCV endocytosis directly by the quantum dots labeled viron infection tactic. Further more, we will investigate the crucial signal regulation events during SVCV endocytosis. Our findings will be important for the revealing of the cell entry mechanism of SVCV and the development of broad spectrum drugs against this virus. Our results also will be helpful in elucidating of the initial steps of Rhabdoviridae infection.

SVCV能够引起鲤科鱼类暴发性出血症，严重危害渔业生产，且目前尚无特异性的治疗药物。阻断病毒的细胞内吞过程，对切断病毒传播具有重要意义。然而，目前SVCV细胞内吞途径、入侵动力学过程以及入侵过程的信号调节机制均尚无报道，有待深入研究。前期，我们利用制备的SVCV M蛋白特异性抗体并结合药物抑制实验初步研究表明SVCV是通过网格蛋白依赖的内吞途径和巨胞饮途径进入细胞的，并且内吞过程依赖于细胞骨架蛋白微丝和微管。本项目拟进一步利用细胞内吞各种途径特异性抑制剂、骨架蛋白解聚剂、内吞关键蛋白突变体、小RNA干扰等手段并结合量子点标记技术，研究SVCV细胞入侵的内吞途径，探索SVCV入侵过程中与细胞骨架蛋白的相互作用关系，揭示SVCV细胞内吞的动态过程，并阐明SVCV细胞内吞的信号调控网络。本项目结果将为SVCV入侵机制的阐明提供系统的实验依据，并为广谱抗病毒药物的研发奠定重要的理论基础。

病毒的细胞入侵是其复制循环的关键步骤，然而，SVCV细胞内吞途径及入侵过程的信号调节机制均尚未揭示。本项目综合利用特异性抑制剂阻断、电镜观察、免疫荧光和实时荧光定量PCR等技术手段，研究发现SVCV是通过网格蛋白介导的内吞途径和巨胞饮途径入侵草鱼卵巢细胞的，并且内吞过程依赖于细胞发动蛋白II、细胞骨架蛋白微管和微丝以及低pH环境。进一步研究发现SVCV入侵过程依赖于Pak1和PKC信号通路的激活，而受到PI3K信号通路的负向调控。此外，本项目成功制备了量子点标记的SVCV病毒颗粒（QD-SVCV），继而实时示踪观察了SVCV的细胞內吞过程，发现其细胞入侵5分钟内即可完成，并进一步获得了QD-SVCV细胞感染的动态行为影像。本项目还对SVCV入侵过程中所识别的细胞表面分子进行了初步研究，结果发现G蛋白可以结合细胞表面非肌肉肌球蛋白Myh10。本项目进一步研究了Myh10同源蛋白Myh9在IPNV感染中的功能，结果发现Myh9在IPNV的细胞入侵及传播中均发挥了重要作用。本项目研究结果首次揭示了SVCV的细胞入侵途径及入侵过程中所依赖的信号分子及其调控网络，为抗SVCV药物的研发奠定了重要的理论基础。