SARS-CoV、EV71与干扰素系统的相互作用

【作者】 徐春娥；

【导师】 陈薇； 侯利华；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2009， 博士

【摘要】 第一部分SARS-CoV与干扰素系统的相互作用本部分对不同干扰素抑制SARS-CoV的作用进行了比较,并对干扰素的抗SARS-CoV作用途径进行了初步探索;研究了SARS-CoV部分辅助蛋白和非结构蛋白对IFN-β启动子和ISG54启动子的作用。在VeroE6细胞和Caco-2两种细胞系中通过观察CPE确定了干扰素抑制SARS-CoV复制的作用,通过计算IFN-β、IFN-ω和IFN-α的EC50得到结论:IFN-β与IFN-ω的作用相似,但二者均大于IFN-α。使用定量PCR方法对不同干扰素保护后的细胞病毒拷贝数进行了检测,发现IFN-β和IFN-ω作用后,细胞中的病毒拷贝数明显降低,IFN-α作用后的降低幅度低于IFN-β和IFN-ω。干扰素抑制SARS-CoV作用是通过其信号转导途径、最后通过效应分子的表达来发挥作用。干扰素信号转导通路涉及的分子很多,本研究使用PCR Array高通量法对这些信号分子进行筛选,以期得到具有显著变化的分子并验证其作用。PCR Array结果发现许多分子都出现了表达上调,包括干扰素相关基因、干扰素受体、干扰素调节因子和干扰素诱导蛋白,其中部分干扰素诱导蛋白的上调达到了千倍,这表明在SARS-CoV感染的Caco-2细胞中,干扰素通过受体表达上调、信号转导调节因子的变化最终通过干扰素效应分子的表达增加而发挥抑制SARS-CoV复制的作用。对PKR、OAS和MxA三种干扰素抗病毒途径的研究发现,IFN-β、IFN-ω和IFN-α预处理的细胞中,OAS的表达都出现了近20倍的上调,而PKR和MxA的表达没有出现明显上调。推测OAS途径可能是干扰素发挥抗病毒作用的途径之一。通过外源表达PKR、OAS和MxA蛋白,发现转染OAS的细胞中SARS-CoV的病毒拷贝数出现明显下降,低于MxA和PKR,说明OAS在干扰素抑制SARS-CoV复制的过程中发挥了一定的作用。干扰素能够抑制SARS-CoV复制,而SARS-CoV也以特有的模式来干扰干扰素的诱导产生,从而逃避干扰素效应分子的抑制作用。从SARS-CoV的非结构蛋白中筛选对IFN-β启动子和ISG54启动子具有抑制作用的分子,发现NSP6和ORF8a对IFN-β启动子和ISG54启动子具有抑制作用,抑制率达50%以上。3a和7a蛋白能够有效增强IFN-β启动子启动的荧光素酶的表达。第二部分EV71与干扰素系统的相互作用EV71是一种比较常见的肠道病毒,多数人感染EV71后,不会有任何症状,只有极少数人会发病,而发病者80%以上是5岁以下儿童。干扰素是治疗病毒感染的常用药物,深入研究干扰素与EV71的相互作用对于指导临床应用很有必要。首先对病毒株进行了基因组测序、亚型鉴定,基因组测序表明本实验中所使用EV71株基因组除3’端外含7324个核苷酸,基因组编码区起自5’端第743位碱基,止于7324位碱基。本株与2008年安徽阜阳流行的其它几个病毒株相比,同源性都在97%以上。与2007年流行的518-01F/SD/CHN/07株的同源性达到99%,病毒VP1基因未发生明显变异,在进化上属C4基因亚型。在Vero E6细胞中比较了先加干扰素再感染病毒和病毒感染后再加干扰素两种处理方式之间的干扰素抗病毒的活性差异,分别从CPE、结晶紫染色、定量PCR、TCID50和细胞形态学观察等方面验证了IFN-β和IFN-ω抑制EV71复制的作用。随后对EV71感染后干扰素不同加入时间的细胞保护作用进行了探索,在EV71感染后6h以内加入干扰素可以有效地抑制病毒复制,而在病毒感染48h后再加入干扰素则对细胞没有保护作用。在EV71感染后,干扰素加入的时间越早对细胞的保护作用越强。接下来研究了EV71诱导干扰素系统发挥作用的能力,发现EV71不能诱导IFN-β和ISG54启动子的表达。IRF3是IFN-β表达调节的重要转录因子,本研究中与对照病毒NDV相比,EV71不能诱导IRF3二聚体化及磷酸化,免疫荧光结果也表明EV71对细胞内IRF3的核转移没有促进作用,说明EV71通过抑制IRF3的激活来阻止干扰素系统的激活。EV71仅有一个开放阅读框,编码VP1、VP2、VP3和VP4四个病毒外壳蛋白和2A、2B、2C、3A、3B、3C、3D七个非结构蛋白。为了说明这些病毒蛋白对干扰素信号通路的影响,研究了它们对IFN-β和ISG54启动子的作用,发现单独的病毒蛋白均没有明显地诱导或抑制IFN-β和ISG54启动子的作用。更多还原

【Abstract】 SectionⅠ: Some exploration on the interaction of SARS-Cronavirus with the interferon systemThe first aim of this study is to compare the antiviral activity of different interferons and explore the anti-SARS-CoV effector pathways of interferons. The second is to study the inhibition effects of some accessory proteins and nonstructural proteins of SARS-CoV on the IFN-β’s promoter and ISG’s promoter.In this report three type I IFNs (IFN-α, -βand -ω) showed different antiviral effects. IFN-βwas a more potent inhibitor of SARS-CoV than IFN-αand -ω. In PCR array analysis, there was no great difference in genes expression profiles among SARS-CoV-infected cells treated with different IFNs (IFN-α, -βand -ω). Some genes related to IFNs expressed differentially in SARS-CoV-infected cells with IFNs treatment compared to without IFNs treatment. OAS1 mRNA increased approximately 20 fold and was further evaluated about its antiviral effect against SARS-CoV in transient transfection assay. Expressions of several interferon-inducible genes, G1P3, IFI27, IFI44 and IFI44L, were increased dramatically, which maybe were related with interferon antiviral effect against SARS-CoV.The anti-SARS-CoV activity of IFN-βhad been verified. So the IFN effector proteins were studied to elucidate their anti- SARS-CoV effect.There are three effect pathways which have been verified, including PKR, 2’-5’OAS and MxA pathway. So the recombinant plsmids containing the three interested genes fragments respectively were constructed and correspond plasmids were transfected instantly into the Vero E6 cells. The anti- SARS-CoV activity was measured by Real-Time PCR. As we expected that the viral copies of cells expressing OAS protein was obviously reduced compared with that of the other two effector proteins. So we can figure out that OAS-1 is one of effetor pathways with which Type I IFNs are against SARS-CoV.The interaction of some SARS-CoV accessory and non-structural proteins with IFN-βpromoter was explored, including NSP6, NSP7, NSP8, ORF3b, ORF7b, ORF 6, UK6, ORF8a, ORF8b, ORF9b and NSP1. The results showed that ORF3a and ORF7a could activate the IFN-βpromoter, but NSP6 and ORF8a could significantly inhibit the expression of luciferase promoted by IFN-βpromoter or by ISG54 promoter. SectionⅡ: Some exploration on the interaction of EV71 with the interferon systemEnterovirus 71 (EV71) belongs to enterovirus. Most people infected with EV71 don’t show any symptoms, only very few will come on, especially the children under 5 years of age. IFNs are used in the anti-virus therapy and can function effectively. So the interaction of EV71 with IFNs is under investigation.The complete genome of EV71 was sequenced and the genotype was identified. The complete genome size is 7324 nt besides the 3’UTR and translated from the 743 site of the 5’UTR, terminated at the 7324 nt site. This isolates have the 97 percent in common with several strains prevalent in Fuyang of Anhui province.VP1-based comparison with EV71 strains showed this strains have the 99% homologous with the 518-01F/SD /CHN / 07 strain prevailed in 2007 and this isolate belong to genogroup C4 .The anti-EV71 activity of IFN-βand IFN-ωwas evaluated in Vero E6 cells by use of CPE, crystal staining, Q-PCR, TCID50 and cell morphological observation. IFN could inhibit EV71’s replication when either added 24h before infection or added 6h after infection. The eralier IFN is added after EV71’s infection, the better it works.EV71 was found not to avtivate IFN-βand ISG54 promoters. IRF3, as an essential transcriptional molecular in IFN-β’s expression, was analyzed. EV71 could not induce IRF-3 activation such as hyperphosphorylation, homodimer formation and nuclear translocation compared with the IFN-inducing control virus NDV. Our data suggest that EV71 appear to block IRF-3 activation in order to escape activation of the IFN system.更多还原