GCRV 感染动态示踪及进入细胞途径研究

To understand how grass carp reovirus（GCRV）enter into host cells during infection,a dynamic tracing of the infectious viral particle entering into living cell and its molecular pathogenesis will be investigated. In this proposed study，quantum dots (QDs), a fluorescent semiconductor nanocrystals with a narrow emission spectrum and excellent photostability, will be used for labeling purified GCRV in vitro. After obtaining QDs labeled GCRV（GCRV-QDs ）, the infectivity and replication properties between GCRV-QDs and native GCRV will be compared by using one step growth curve and qPCR assays, and dynamic tracing image of GCRV-QDs in infected living cells will be also performed. In addition, disassembly and conformational changes of outer capsid VP5 and VP7 proteins during early stage of viral infection will be detected using immunofluorescence and immunoblotting. Moreover, inhibitors that are related to various cellular entry pathways will be investigated by drug treatment experiments or small interfering RNAs(siRNAs) to understand the refined dynamic behavior of GCRV during cell entry. The study will not only provide a solid and lively image to visualize the dynamic tracing of GCRV infection at early stage, but also will reveal entry pathway and the molecular pathogenesis of GCRV or dsRNA viruses during entry into cells.

为探讨草鱼呼肠孤病毒（GCRV）在侵染过程中进入细胞的分子机制，本项目拟依据纳米材料量子点独特的荧光性质及超强的光稳定性等特点，采用量子点对GCRV进行体外分子标记，构建量子点标记的GCRV颗粒（GCRV-QDs）。应用实时定量PCR等方法，比较GCRV-QDs与GCRV颗粒的感染特性，继而进行GCRV-QDs感染细胞实时动态示踪，获得GCRV-QDs感染细胞的动态行为影像。利用已制备的VP5、VP7抗体，检测病毒侵染细胞过程中VP5和VP7蛋白复合物的解聚与构象变异。此外，通过细胞因子药物抑制实验，结合荧光标记网格蛋白及其它膜转运蛋白、siRNA干扰等分子生物学技术，研究GCRV-DQs入侵细胞的精细行为，探明病毒入侵细胞的动态行为方式。该研究对于揭示GCRV及dsRNA病毒感染的分子机制具有十分重要的理论与实践意义。

草鱼呼肠孤病毒（Grass carp reovirus, GCRV）被认为是具有强致病性的水生动物呼肠孤病毒，但对其致病机理知之甚少。本研究采用量子点（quantum dots, QDs）标记的GCRV原位示踪并结合多种生物化学及分析生物学方法，对GCRV进入细胞的有效途径进行了研究。结果表明，MβCD (methyl-β-cyclodextrin) 和 nystatin能有效抑制GCRV进入细胞。此外，采用抑制小窝感染途径的药物genistein处理细胞，GCRV进入细胞呈现明显减少。随后的活细胞原位实时示踪实验检测到QDs标记的GCRV与小窝蛋白-1(caveolin-1)具有共定位；进一步采用构建的显性抑制突变质粒 (caveolin-1 Y14F)转染细胞，能有效抑制病毒的复制。然而，采用网格介导的内吞途径及大胞饮抑制剂chlorpromazine (CPZ) ,hyperosmotic sucrose and blebbistatin,未检测到对病毒进入细胞的影响。上述结果表明，水生呼肠孤病毒起始有效的感染可能通过小窝介导的内吞途径，而非网格与大胞饮途径。在上述基础上，采用内吞体抑制剂(ammonium chloride, chloroquine, and bafilomycin A1)处理细胞，GCRV感染受到明显抑制，表明GCRV感染依赖于酸性内吞体。进一步采用细胞骨架抑制剂nocodazole和 cytochalasin D 处理细胞，发现仅nocodazole对病毒入侵产生明显的影响。综上，GCRV利用小窝介导的内吞体与微管依赖的内吞途径有效感染宿主细胞。