Over-expression of rabies virus G protein and its inhibitory effect on the virus in neuroblastoma cells
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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:ObjectiveTo 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.
MethodThe 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.
ResultHep-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.
ConclusionThis 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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Keywords:
- rabies virus /
- glycoprotein /
- IFN-β pathway /
- interferon-β /
- virus titer
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图 1 Hep-dG感染NA细胞激活IFN-β信号通路
a: Hep-dG感染增强IFN-β基因的表达水平，图中“*”、“**”和“***”分别表示直线两端所对应的样本差异达0.05、0.01和0.001的显著水平(t检验)；b:Hep-dG感染刺激STAT1的表达和激活，1：空白对照，2：rHep-Flury，3：Hep-dG
Figure 1. Hep-dG infection activated the IFN-β signaling pathway in NA cells
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图 2 狂犬病病毒吸附和入侵NA细胞的分析
a：细胞表面吸附狂犬病病毒的分析；b：狂犬病病毒入侵NA细胞的分析，各图中“*”和“**”分别表示直线两端所对应的样本差异达0.05和0.01的显著水平(t检验)
Figure 2. Analyses of RABVs adsorbing and invading NA cells under different infection multiplicities
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图 3 狂犬病病毒感染对Leader RNA、vRNA和RIG-I基因表达的影响
a：Leader RNA的表达，b：vRNA的表达，c：RIG-I基因的表达；各图中“**”和“***”分别表示直线两端所对应的样本差异达0.01和0.001的显著水平(t检验)
Figure 3. Effects of rabies virus infections on expressions of Leader RNA，vRNA and RIG-Igene
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图 4 基于最小自由能的狂犬病病毒rHep-Flury和Hep-dG Leader RNA的二级结构
Figure 4. The secondary structure of rabies viruses rHep-Flury and Hep-dG Leader RNA based on the minimum free energy
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图 5 G蛋白的真核表达对NA细胞中IFN-β转录的影响
a：pH-G、pH-M和pH-GFP的荧光表达；b：IFN-β基因的表达，CK：阴性对照，1：5.0 μg的pH-G，2：2.5 μg的pH-G，3：5.0 μg的pH-M，4：2.5 μg的pH-M，5：阳性对照，图中“*”表示直线两端所对应的样本达0.05的显著水平(t检验)
Figure 5. Effect of G protein eukaryotic expression on IFN-β transcription in NA cell
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图 6 阻断IFN-β通路对狂犬病病毒复制的影响
a.抗体阻断IFN-β的表达，CK：空白对照，1：rHep-Flury，2：Hep-dG，3：rHep-Flury+IgG，4：Hep-dG+IgG，5：rHep-Flury+Anti，6：Hep-dG+Anti，图中“*”和“**”分别表示直线两端所对应的样本达0.05和0.01的显著水平(t检验)；b.病毒滴度的检测，1：rHep-Flury，2：Hep-dG，3：rHep-Flury+IgG，4：Hep-dG+IgG，5：rHep-Flury+Anti，6：Hep-dG+Anti
Figure 6. Effect of blocking IFN-β pathway on rabies virus replication
表 1 荧光定量PCR检测引物
Table 1 Primers used for fluorescence quantitative PCR detection
被检基因 引物序列 引物名称 产物长度/bp Leader RNA Leader-F 5'-CCAGATGCTTGGCGTCCT-3' 67 Leader-R 5'-ACGCTTAACAACAAAACC-3' vRNA vRNA-F 5'-GGAAAAGGGACATTTGAAAGAA-3' 130 vRNA-R 5'-AGTCCTCGTCATCGGAGTTGAC-3' RIG-I RIGI-F 5'-GCAAGTGCTTCCTCCTGACC-3' 142 RIGI-R 5'-ATGCGGTGAACCGTCTTTCC-3' GAPDH GAPDH-F 5'-AGAGTGTTTCCTCGTCCCGT-3' 199 GAPDH-R 5'-CTGTGCCGTTGAATTTGCCG-3' 
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