NTPase调控杯状病毒复制及致病的分子机制

Norovirus (NV) is an important pathogen casing diarrhea in human. But its pathogenisis remains unknown due to the lack of highly efficient in vitro culture system. Feline calicivirus (FCV) and murine norovirus (MNV) are able to be cultured in vitro. MNV infection could not produce the disease as well as significant clinic symptoms, but FCV infection could cause severe respiratory and digestive diseases, suggesting FCV infection may be an ideal model for the research of calicivirus biological property. The infectious clones of two FCV strains with significant variance in viral replication and pathogenicity were constructed in our previous study. Using this system, we found that NTPase affects viral replication and virulence. To explore the mechanism about how NTPase regulates the replication and pathogenicity of caliciviruses, the virulence determinant animo acid would be identified by comparision of the recombiant viruses present with a single amino acid mutation. The role of the virulence determinant animo acid in viral invasion, replication and budding will be analyzed. Next, the cDNA library will be construced, and the host genes associated with NTPase and affecting viral replication will be identified through gene knockout and overexpression methods. Finnally, the asssociation of these host genes with the NTPase of NV, Rabbit Haemorrhagic Disease virus and other caliciviruses would be analyzed. Whether these host genes can affect viral packaging in vitro would be further analyzed to explain the molecular machanism about how NTPase regulates the replication and pathogenicity of caliciviruses.

诺如病毒（NV）是引起人病毒性腹泻的重要病原，但缺乏高效体外培养系统，其致病机制不清楚。猫杯状病毒（FCV）和鼠诺如病毒 (MNV) 可以体外培养，但MNV对小鼠并不致病，而感染FCV可引起猫严重的呼吸道和消化道疾病，因此FCV是研究杯状病毒致病机制的最佳模型。项目前期构建了体内外复制及致病性差异明显的FCV毒株反向遗传系统，发现NTPase决定FCV复制及毒力。为解析NTPase调控杯状病毒致病的分子机制，将NTPase单一氨基酸位点突变，筛选调控病毒复制关键位点，分析该位点在病毒入侵、复制及出芽中的作用；其次构建细胞cDNA文库，筛选与NTPase互作宿主基因，通过基因敲除及过表达技术筛选影响病毒复制的宿主基因，并验证这些宿主基因与NV、兔出血热病毒等杯状病毒NTPase的相互作用及对病毒体外包装的影响，解析NTPase调控杯状病毒复制机制，分析该基因是否可成为病毒防控靶点。

FCV属于杯状病毒科，是该科少有的能够在细胞培养的病毒。一旦发生感染，FCV可以在猫群中持续存在，但是病毒如何逃逸宿主先天免疫反应的分子机制仍然未知。.本研究发现，FCV强毒株2280能够拮抗IFN-β抗病毒作用，进一步解析了其潜在的分子机制。机制研究表明，FCV 2280感染可以通过诱导IFNAR1 mRNA的降解抑制细胞中I型干扰素受体IFNAR1的表达，从而抑制下游接头分子的磷酸化。通过qPCR和Northern blot方法发现FCV 2280株 p30蛋白过表达可以下调IFNAR1 mRNA的表达而弱毒株F9 p30不能抑制；体外生化实验证明2280 p30可以直接降解IFNAR1 RNA。.进一步研究，将2280 p30和F9 p30进行互相替换，构建嵌合病毒—rFCV 2280-F9 p30和rFCV F9-2280 p30。与亲本病毒感染相比，rFCV 2280-F9 p30感染细胞后在降低IFNAR1 mRNA表达能力被削弱，而rFCV F9-2280 p30的活性增强，能够明显下调IFNAR1 mRNA表达。动物实验结果显示，F9 p30的替换降低了2280的致病力，而2280 p30的表达增强了F9毒株感染家猫后的毒力。.通过RNA-seq和同位素技术也进一步证明FCV感染细胞后能够导致宿主基因关闭。除p30外，病毒的PP蛋白（Polymerase-Proteinase）也同样能够直接降解宿主mRNA，导致宿主基因家族mRNA下调表达以上这些数据表明，FCV 2280株p30蛋白和PP蛋白可以直接选择性降解宿主mRNA，阻断I型干扰素信号通路及其他抗病毒应答相关信号通路的激活，从而逃逸宿主的抗病毒应答，揭示了FCV感染细胞后快速复制的分子机制。