The different regulatory effect of glycoproteins from virulent and attenuated strain of rabies virus on type I interferon signaling pathway
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摘要:目的
狂犬病(Rabies)是由狂犬病病毒(Rabies virus，RABV)引起的一种高致死性人兽共患传染病。I型干扰素(IFN-I)通路在抵抗RABV感染中发挥重要作用。RABV可通过磷蛋白及核蛋白的功能逃逸IFN-I的抗病毒作用，本研究旨在探讨对RABV的致病性有重要影响的糖蛋白(Glycoprotein，G)在调节IFN-I通路方面扮演怎样的角色。
方法将RABV弱毒株Hep-Flury的G基因替换成致病株CVS-11的G基因，拯救得到重组病毒HepG，分析Hep-Flury、CVS-11和HepG这3种毒株在体内和体外感染对IFN-I通路激活和调控的差别，比较它们在神经细胞中对抗IFN-I抗病毒作用的差异性。
结果替换了CVS-11的G基因后，重组病毒HepG致病力增强，能够100%致死小鼠，在鼠脑中的增殖水平显著高于亲本株Hep-Flury。在感染鼠脑早期及体外神经细胞时，弱毒株Hep-Flury能够较快地激活IFN-I通路相关基因的表达，重组病毒HepG的激活能力介于Hep-Flury和CVS-11之间。利用Poly(I:C)激活神经细胞的IFN-I通路后，Hep-Flury的增殖被显著抑制，CVS-11和HepG的复制几乎不受影响，表现出一定的抵抗能力。
结论RABV的G蛋白在调节和抵抗IFN-I通路方面发挥重要功能，为进一步探究RABV致病毒株的G蛋白如何协助病毒在中枢神经系统中逃逸IFN-I提供了线索和依据。
Abstract:ObjectiveRabies is a highly lethal zoonotic infectious disease caused by rabies virus (RABV). Type I interferon (IFN-I) pathway plays an important role in resisting RABV infection. RABV can escape the antiviral effect of IFN-I through the function of its phosphoprotein and nucleoprotein. The aim of the study was to investigate the role of glycoprotein (G), which has an important impact on the pathogenicity of RABV, in regulating IFN-I pathway needs more comprehensive exploration.
MethodThis study replaced the G gene of the RABV attenuated strain Hep-Flury with the G gene of the pathogenic strain CVS-11 to rescue and acquire the recombinant virus HepG. We analyzed the differences in the activation and regulation of IFN-I pathway in vivo and in vitro infected with Hep-Flury, CVS-11 and HepG, and compared the differences of these virus strains in fighting against antiviral effect of IFN-I in nerve cells.
ResultAfter replacing G gene, the recombinant virus HepG had enhanced pathogenicity, was able to kill 100% of mice and the proliferation level in the mouse brain was significantly higher than that of the parental strain Hep-Flury. While infecting mouse brain early and in vitro neuronal cells, the attenuated strain Hep-Flury was able to activate the expression of IFN-I pathway-related genes faster, and the activation ability of HepG was between that of Hep-Flury and CVS-11. After activation of the IFN-I pathway in neuronal cells using Poly(I:C), the proliferation of Hep-Flury was significantly inhibited, and the replication of CVS-11 and HepG was almost unaffected, showing some resistance.
ConclusionG protein of RABV plays an important role in regulating and resisting the IFN-I pathway, providing the clue and evidence for further exploring how the G protein of RABV pathogenic strains helps the virus escape IFN-I pathway in the central nervous system.
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Keywords:
- Rabies /
- Rabies virus /
- Type I interferon /
- Glycoprotein /
- Immune evasion
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图 2 构建重组病毒HepG的扩增片段
M：DNA marker DL10000；1、2：C区段基因；3、4：L片段基因；5、6：CVS-11 G基因
Figure 2. Amplified fragments for construction of recombinant virus HepG
M: DNA marker DL10000; 1, 2: C-segment gene; 3, 4: L-segment gene; 5, 6: CVS-11 G gene
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图 4 3种RABV毒株感染的KM小鼠的体质量变化
Figure 4. Body weight changes of KM mice infected with three RABV strains
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图 5 RABV感染鼠脑后病毒载量和病毒基因转录水平变化
“*”“**”“***”分别表示在 P<0.05、 P<0.01、 P<0.001水平差异显著(t检验)
Figure 5. Changes of viral load and viral gene transcription in the brains of mice infected with RABV
“*”, “**” and “***” indicate significant differences at P <0.05, P <0.01 and P <0.001 levels, respectively (t test)
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图 6 RABV感染鼠脑后IFN-I通路相关基因表达分析
“*”“**”“***”分别表示在 P<0.05、 P<0.01、 P<0.001水平差异显著（t检验）
Figure 6. Expression analysis of IFN-I pathway related genes in the brains of mice infected with RABV
“*”, “**” and “***” indicate significant differences at P <0.05, P <0.01 and P <0.001 levels, respectively (t test)
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图 7 RABV感染鼠脑后不同IFN基因表达分析
“*”“**”“***”分别表示在 P<0.05、 P<0.01、 P<0.001水平差异显著（t检验）
Figure 7. Expression analysis of IFN genes in the brains of mice infected with RABV
“*”, “**” and “***” indicate significant differences at P <0.05, P <0.01 and P <0.001 levels, respectively (t test)
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图 8 不同RABV毒株感染Neuro-2a细胞的一步生长曲线
“*”“**”分别表示CVS-11/HepG与Hep-Flury在P<0.05和P<0.01水平差异显著（t检验）
Figure 8. One-step growth curve of different RABVs in Neuro-2a
“*” and “**” indicate significant differences between CVS-11/HepG and Hep-Flury at P<0.05 and P<0.01 levels, respectively (t test)
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图 9 RABV感染Neuro-2a细胞对IFN-I通路上游基因表达的影响
“*”“**”“***”分别表示在 P<0.05、 P<0.01、 P<0.001水平差异显著（t检验）
Figure 9. Effect of RABV infection on expression of upstream genes of IFN-I pathway in Neuro-2a cells
“*”, “**” and “***” indicate significant differences at P <0.05, P <0.01 and P <0.001 levels, respectively (t test)
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图 10 RABV感染Neuro-2a细胞对IFN-I通路下游基因表达的影响
“*”“**”“***”分别表示在 P<0.05、 P<0.01、 P<0.001水平差异显著（t检验）
Figure 10. Effect of RABV infection on expression of downstream genes of IFN-I pathway in Neuro-2a cells
“*”, “**” and “***” indicate significant differences at P <0.05, P <0.01 and P <0.001 levels, respectively (t test)
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图 11 Poly(I:C)处理体外神经细胞对RABV复制的影响
“*”表示相同RABV毒株感染Poly(I:C)处理和未处理细胞在P<0.05水平差异显著(t检验)
Figure 11. Effect of Poly(I:C) treatment on the replication of RABV for in vitro nerve cells
“*” indicates significant differences between Poly(I:C) treated and untreated cells infected with the same RABV strain at P<0.05 level (t test)
表 1 HepG构建的引物序列
Table 1 Primer sequences for construction of HepG
基因片段
Gene segment引物序列(5′→3′)
Primer sequenceHEP-L F: AGGCCGGTCATCCTTTTGACACCTCAAGTCCAGA R: ATTTTAGCATGTACAGGCTTTCATTAATGTCCGGC HEP-C F: GCCTGTACATGCTAAAATTCTTGTAYGATGCATCTTG R: CTTTCCTTAAGTCTTTTGAGGGATGTTAATAGTTTT CVS-G F: CAAAAGACTTAAGGAAAGATGGTTCCTCAGGTTCTT R: CAAAAGGATGACCGGCCTTCACAGTCTGATCTCACCT 
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表 2 RT-qPCR检测引物
Table 2 Primers used for RT-qPCR
基因
Gene上游引物序列(5′→3′)
Forward primer sequence下游引物序列(5′→3′)
Reverse primer sequenceRABV genome GACAGCGTCAATTGCAAAGCAAAAAT GGGTACTTGTACTCATATTGATCCACGAT N GCACTGGCAGATGACGGAAC TCGGCGAATGAGTTTGGACG Leader RNA TGTAGGGGTGTTACATTTTT ACGCTTAACAACAAAACC Cxcl10 TCCATATCGATGACGGGCCA CAACACGTGGGCAGGATAGG Dhx58 CCAGAGCACACACATGACCC CCATAGCGCACCACCACATT IFN-α4 AGCCTGTGTGATGCAGGAACC CAGCAAGTTGGTTGAGGAAGAG IFN-κ TGGAGTTGGGCAAGTATTTCTTC GGACTTGGAAAATATAATGAAACATCTTC IFN-β ACCTACAGGGCGGACTTCAA ACACTGTCTGCTGGTGGAGT IFN-γ TCAGCAACAGCAAGGCGAAA ATTGAATGCTTGGCGCTGGA IL6 TGCCTTCTTGGGACTGATGC GACAGGTCTGTTGGGAGTGGT IRF3 TCAGGATCCCGTGGAAGCAT TGACACGTCCGGCTTATCCT IRF7 ACTGGCTATTGGGGAGGTC ACGACCGAAATGCTTCCAGG ISG15 GACGCAGACTGTAGACACGC TTAGGCCATACTCCCCCAGC MDA5 GAGCACCTACGCACTTTCCC CCACCGTCGTAGCGATAAGC MyD88 TCATGTTCTCCATACCCTTGGT AAACTGCGAGTGGGGTCAG RIG-I CCTTGATTGCCCTGATGTTG ATTTCTCCCTGTCCCTCCAA RNF125 GGCGCACATAAGGACCTGTG TCATCCAGTTCCCGCTGACA TLR3 GTGAGATACAACGTAGCTGACTG TCCTGCATCCAAGATAGCAAGT TLR7 TCTTTGGGTTTCGATGGTTTCC GCAGTCCACGATCACATGGG TLR9 ATGGTTCTCCGTCGAAGGACT GAGGCTTCAGCTCACAGGG GAPDH AGAGTGTTTCCTCGTCCCGT CTGTGCCGTTGAATTTGCCG
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