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    阳佑天, 张琼, 张博越, 等. 狂犬病病毒G蛋白的过表达及对病毒的抑制[J]. 华南农业大学学报, 2018, 39(6): 10-17. doi: 10.7671/j.issn.1001-411X.2018.06.003
    引用本文: 阳佑天, 张琼, 张博越, 等. 狂犬病病毒G蛋白的过表达及对病毒的抑制[J]. 华南农业大学学报, 2018, 39(6): 10-17. doi: 10.7671/j.issn.1001-411X.2018.06.003
    shu
    YANG Youtian, ZHANG Qiong, ZHANG Boyue, LIU Wenjun, LUO Yongwen, ZHAO Jing, MEI Mingzhu, ZHANG Ying, LUO Jun, GUO Xiaofeng. Over-expression of rabies virus G protein and its inhibitory effect on the virus in neuroblastoma cells[J]. Journal of South China Agricultural University, 2018, 39(6): 10-17. DOI: 10.7671/j.issn.1001-411X.2018.06.003
    Citation: YANG Youtian, ZHANG Qiong, ZHANG Boyue, LIU Wenjun, LUO Yongwen, ZHAO Jing, MEI Mingzhu, ZHANG Ying, LUO Jun, GUO Xiaofeng. Over-expression of rabies virus G protein and its inhibitory effect on the virus in neuroblastoma cells[J]. Journal of South China Agricultural University, 2018, 39(6): 10-17. DOI: 10.7671/j.issn.1001-411X.2018.06.003
    shu

    狂犬病病毒G蛋白的过表达及对病毒的抑制

    Over-expression of rabies virus G protein and its inhibitory effect on the virus in neuroblastoma cells

      摘要
    • 摘要:
      目的  探究G蛋白在狂犬病病毒(Rabies virus，RABV)复制中的作用，以揭示携带双G基因的重组RABV的Hep-dG与亲代毒株rHep-Flury在神经母细胞瘤(NA)细胞中滴度差异的原因，为RABV致病机制的研究奠定基础。
      方法  通过病毒吸附、入侵、荧光定量PCR、Western-blot以及中和抗体阻断等试验，检测G蛋白过表达对IFN-β以及相关因子转录的影响。
      结果  Hep-dG感染能显著上调NA细胞中IFN-β mRNA的表达，激活了下游因子STAT1的表达与磷酸化，在较低的感染复数(MOI=0.01)下，Hep-dG感染后24 h即可显著促进IFN-β 基因的表达，36 h达到最高水平(P<0.001)。该病毒进入细胞后，产生了更多的病毒Leader RNA和RIG-I mRNA，且与IFN-β mRNA的表达高度一致。抗体阻断IFN-β后，Hep-dG在NA细胞中的病毒滴度显著上升(P<0.01)，约为阻断前的7.9倍，且与亲代毒株rHep-Flury无显著差异。与阴性对照比较，5 μg的pH-G质粒转染能刺激IFN-β的转录(P<0.05)，表明真核表达RABV G蛋白能在一定程度上刺激IFN-β的转录。
      结论  本研究初步揭示了G蛋白激活先天性免疫应答的原因和作用。RABV G蛋白的过表达，通过促进病毒Leader RNA的转录，激活了RIG-I介导的IFN-β通路，进而抑制了Hep-dG在NA细胞的繁殖。G蛋白的过表达也对干扰素通路起到一定的作用。

       

      Abstract:
      Objective  To explore the role of G protein in rabies virus (RABV) replication, reveal the reason for the difference of virus titer in neuroblastoma (NA) cells between the recombinant RABV Hep-dG with dual copy of G gene and the parental strain rHep-Flury, and lay a foundation for the study of RABV pathogenesis.
      Method  The effects of G protein over-expression on transcriptions ofIFN-β and related factors were examined by the virus binding assay, virus entry assay, fluorescence quantitative PCR, Western-blot and neutralizing antibody blocking assay.
      Result  Hep-dG infection significantly increased the expression of IFN-β mRNA and activated the expression of the downstream factor STAT1 in NA cells. Under the low multiplicity of infection (MOI=0.01), the expression of IFN-β gene significantly increased at 24 h after Hep-dG infection and reached the highest level at 36 h. After the virus entered the cells, there were more viral Leader RNA and RIG-I mRNA, which were highly consistent with the expression of IFN-β mRNA. The block of IFN-β expression by neutralizing antibody in NA cells significantly increased the virus titer of Hep-dG in cell culture supernatant(P<0.01), which was 7.9 times before blocking. Meanwhile the virus titer of Hep-dG had no significant difference with the parental strain rHep-Flury. Compared with the negative control, transfection of 5 μg pH-G plasmid could stimulate the transcription ofIFN-β(P<0.05), which showed that eukaryotic expression of RABV G protein could stimulateIFN-β transcription to a certain extent.
      Conclusion  This study preliminarily reveals the cause and role of G protein in activating innate immune response. Over-expression of RABV G protein activates the RIG-I-mediated IFN-β pathway by promoting transcription of the viral Leader RNA, which in turn inhibits Hep-dG replication in NA cells and finally results in the lower virus titer in NA cells.

       

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