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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[1] 王朝, 周明, 傅振芳, 等. 狂犬病病毒逃逸宿主天然免疫反应的研究进展[J]. 生命科学, 2017(3): 237-244. [2] 张旭, 纪森林, 赵雯, 等. 狂犬病病毒致病机制的最新研究进展[J]. 中国兽医科学, 2018, 48(3): 295-304. [3] MEBATSION T, KONIG M, CONZELMANN K K. Budding of rabies virus particles in the absence of the spike glycoprotein[J]. Cell, 1996, 84(6): 941-951.
[4] MEBATSION T, WEILAND F, CONZELMANN K K. Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G[J]. J Virol, 1999, 73(1): 242-250.
[5] MACFARLAN R I, DIETZSCHOLD B, WIKTOR T J, et al. T cell responses to cleaved rabies virus glycoprotein and to synthetic peptides[J]. J Immunol, 1984, 133(5): 2748-2752.
[6] 汪孟航, 朱洪伟, 何民辉, 等. 狂犬病病毒糖蛋白生物学功能研究进展[J]. 中国畜牧兽医, 2016(12): 3349-3355. [7] FISHER C R, STREICKER D G, SCHNELL M J. The spread and evolution of rabies virus: Conquering new frontiers[J]. Nat Rev Microbiol, 2018, 16(4): 241-255.
[8] LIU X, YANG Y, SUN Z, et al. A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge[J]. PLoS One, 2014, 9(2): e87105.
[9] PREHAUD C, LAY S, DIETZSCHOLD B, et al. Glycoprotein of nonpathogenic rabies viruses is a key determinant of human cell apoptosis[J]. J Virol, 2003, 77(19): 10537-10547.
[10] FABER M, PULMANAUSAHAKUL R, HODAWADEKAR S S, et al. Overexpression of the rabies virus glycoprotein results in enhancement of apoptosis and antiviral immune response[J]. J Virol, 2002, 76(7): 3374-3381.
[11] YANG Y, LIU W, YAN G, et al. iTRAQ protein profile analysis of neuroblastoma (NA) cells infected with the rabies viruses rHep-Flury and Hep-dG[J/OL]. Front Microbiol, 2015, 6: 691. doi: 10.3389/fmicb.2015.00691.
[12] HABJAN M, PENSKI N, SPIEGEL M, et al. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus[J]. J Gen Virol, 2008, 89(9): 2157-2166.
[13] YANG Y, HUANG Y, GNANADURAI C W, et al. The inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader RNA[J]. J Virol, 2015, 89(4): 2157-2169.
[14] WU J, MA C, WANG H, et al. A MyD88-JAK1-STAT1 complex directly induces SOCS-1 expression in macrophages infected with Group A Streptococcus[J]. Cell Mol Immunol, 2015, 12(3): 373-383.
[15] OGINO T, YADAV S P, BANERJEE A K. Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase[J]. Proc Natl Acad Sci USA, 2010, 107(8): 3463-3468.
[16] GERLIER D, LYLES D S. Interplay between innate immunity and negative-strand RNA viruses: Towards a rational model[J]. Microbiol Mol Biol Rev, 2011, 75(3): 468-490.
[17] MOYER S A, ABRAHAM G, ADLER R, et al. Methylated and blocked 5' termini in vesicular stomatitis virus in vivo mRNAs[J]. Cell, 1975, 5(1): 59-67.
[18] BRZOZKA K, FINKE S, CONZELMANN K K. Identification of the rabies virus alpha/beta interferon antagonist: Phosphoprotein P interferes with phosphorylation of interferon regulatory factor 3[J]. J Virol, 2005, 79(12): 7673-7681.
[19] RIEDER M, CONZELMANN K K. Interferon in rabies virus infection[J]. Adv Virus Res, 2011, 79: 91-114.
[20] 王林栋, 张守峰, 刘晔, 等. 基于RIG-I的狂犬病病毒免疫逃逸机制[J]. 中国生物制品学杂志, 2013(10): 1517-1521. [21] VIDY A, CHELBI-ALIX M, BLONDEL D. Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways[J]. J Virol, 2005, 79(22): 14411-14420.
[22] VIDY A, EL B J, CHELBI-ALIX M K, et al. The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1[J]. J Virol, 2007, 81(8): 4255-4263.
[23] PLUMET S, HERSCHKE F, BOURHIS J M, et al. Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response[J]. PLoS One, 2007, 2(3): e279.
[24] FAUL E J, WANJALLA C N, SUTHAR M S, et al. Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling[J]. PLoS Pathog, 2010, 6(7): e1001016.
[25] KATZ I, GUEDES F, FERNANDES E R, et al. Immunological aspects of rabies: A literature review[J]. Arch Virol, 2017, 162(11): 3251-3268.
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