Asia1型口蹄疫病毒RGD基序突变株识别受体的鉴定和比较

The interaction between virus and its cellular receptor initiates a chain of dynamic events that will enable entry of the virus into the cell. In previous studies, we found that two foot and mouth disease viruses harbored alternative receptor binding sites (Arg-Asp-Asp (RDD) or Arg-Ser-Asp (RSD)) instead of the Arg-Gly-Asp (RGD) integrin recognition motif. Using reverse genetics technique, the RDD-, RSD-, and RGD-containing FMD viruses were rescued, and the data showed different pathogenicity and host tropism in mouse, swine and bovine. The objective of this study is to determine what is the molecular basis underlying the virulence and host tropism of FMD viruses, and how the non-RGD FMD viruses recognize the receptor-mediated pathways. These virus-receptor interactions that will provide links to virus tropism and pathogenesis. To achieve: (1) stable cell lines expressing αvβ3，αvβ6，αvβ1 and αvβ8 from mouse, swine and bovine will be established. (2) These established cell lines and the cells only expressed heparan sulfate (HS) will be infected by the above-mentioned FMD viruses and the molecular biological characteristics including plaque forming units (PFU), 50% tissue culture infectious dose (TCID50) and viral replication kinetic curve will be examined and analyzed.（3）The pgsD-677(a CHO cell mutant deficient for heparan sulfate polymerization)will be infected and analyzed by non-RGD FMD viruses.(4) The cell lines expressing different host receptors such as αvβ3，αvβ6，αvβ1, αvβ8 or HS will be infected by same strain. The above-mentioned molecular biological characteristics will be measured. The results will be useful as the molecular basis underlying the virulence and host tropism of the foot-and-mouth disease viruses.

病毒受体结合位点与细胞表面受体结合是启动口蹄疫病毒（FMDV）感染的第一步，决定着病毒感染细胞类型、致病性和宿主嗜性等。前期研究发现，Asia1型FMDV的受体结合位点RGD突变成了RSD或RDD，致其对乳鼠、牛和猪三种宿主都发生较大的致病性差异和宿主嗜性变异。为了阐明RGD突变致其宿主嗜性变异的受体路径，本研究用反向遗传技术制备的含RGD、RSD和RDD基序的毒株平行感染同一宿主的各个单受体（αvβ3、αvβ6、αvβ1、αvβ8和HS）细胞系及整联蛋白与HS受体双阴性细胞系，比较测定这些病毒与受体结合、侵入和复制情况，进一步确定这些毒株生物学特性（PFU、TCID50和病毒复制动力曲线等）的差异，鉴定RGD基序突变株对受体路径的识别和差异。并从宿主的角度，比较研究牛和猪的受体路径对FMDV感染性的影响。为进一步鉴定FMDV第三未知受体和阐明"FMDV宿主嗜性变异"的科学问题奠定基础。

口蹄疫病毒受体结合位点与细胞表面的受体结合启动了病毒感染的第一步，决定了病毒感染的细胞类型和宿主嗜性。为了研究FMDV受体结合位点突变对受体路径识别和利用的影响，本项目利用反向遗传技术，构建了仅受体结合位点RGD突变成RSD、RDD的重组毒株，在稳定表达鼠源整联蛋白受体αvβ3、αvβ1、αvβ6和αvβ8的细胞系上测定并比较了RGD和非RGD基序重组毒及A型、O型等不同毒株的生物学特性差异（PFU，TCID50和病毒复制动力学曲线等），同时，在分别含硫酸乙酰肝素受体和第三受体的细胞系中，分析了RGD和非RGD基序毒株利用第二受体和第三受体的差异。建立了牛源和猪源整联蛋白受体瞬时表达系统，比较了RGD突变毒株对猪源和牛源整联蛋白受体的识别能力。结果表明，RGD基序的突变改变了口蹄疫病毒识别整联蛋白受体的能力，且同一毒株对来自于不同宿主的同一种整联蛋白受体的识别能力不同。同时，鉴定了RGD基序后第9位氨基酸由S突变为D后增强了口蹄疫病毒在细胞和宿主上的致病能力，利用猪源整联蛋白受体的瞬时表达系统比较了突变毒株对整联蛋白受体的识别能力，结果表明，该突变增强了口蹄疫病毒对整联蛋白受体的利用能力，尤其是αvβ6和αvβ8。建立了FMDV利用第三受体的感染模型，开展了第三受体介导的内吞路径的研究，结果表明FMDV利用第三受体入侵宿主细胞可以同时利用网格蛋白和小窝蛋白介导的内吞，在此基础上，还证明了病毒入侵依赖发动蛋白和核内体的酸性环境。本研究揭示了含不同受体结合位点的FMDV识别和利用受体路径的差异对FMDV致病性及宿主嗜性的影响，探明了FMDV入侵宿主细胞的方式，为深入鉴定FMDV利用的未知第三受体提供理论依据。