【作者】 谢立兰；

【导师】 方六荣；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2011， 博士

【摘要】 病毒感染与宿主免疫的机制是当今病毒学研究最重要的前沿领域之一。病毒感染宿主细胞时,细胞表达的模式识别受体(pattern recognition receptors, PRRs)识别和递呈病毒的病原相关分子模式(pathogen-associated molecular patterns, PAMPs),经一系列的信号转导最终诱导I型干扰素(IFN-I)等细胞因子的表达。从感染细胞分泌的IFN-I与细胞膜上IFN-I共有的受体结合后,激活相应的信号转导,诱导其他抗病毒蛋白与免疫调节因子的表达,从而抑制病毒的复制与传播,并激活获得性免疫系统进一步清除入侵的病毒,在机体的抗病毒过程中起重要作用。尽管近年来的研究初步揭示了病毒感染诱导IFN-I产生的过程,但仍有很多未知的问题有待进一步探讨。例如,新发现的识别DNA和RNA的信号蛋白识别病毒并诱导IFN-I的分子机制还不清楚,而细胞中是否还存在其它可以激活IFN-I的蛋白,DNA病毒的识别受体及其介导IFN-I产生的机制是什么等等。因为具有低成本和高相似性等特点,猪的免疫系统逐渐成为人体免疫机制研究的模型。为了揭示病毒感染猪源细胞后诱导IFN-I产生的分子机制,本研究从猪源细胞中克隆了2个参与天然免疫的重要细胞信号分子,并分别以伪狂犬病毒(Pseudorabies Virus, PRV)和猪传染性胃肠炎病毒(Transmissible Gastroenteritis Virus, TGEV)为DNA病毒和RNA病毒的模型对其感染细胞后的天然免疫反应信号通路进行了研究。具体内容如下：1.猪DAI基因的克隆及其功能鉴定DAI(DNA-dependent activator of IFN-regulatory factors)是被发现的第一个介导细胞内dsDNA诱导天然免疫的受体蛋白,但研究发现DAI的作用可能存在细胞特异性和种属特异性。为此根据与人DAI基因有较高同源性的猪基因组序列设计引物,以猪外周血单核细胞的RNA为模板克隆得到猪DAI全长cDNA。序列分析表明,猪DAI开放阅读框全长为1320bp(GenBank收录号：FJ455510),编码439个氨基酸。进一步对推导的氨基酸序列进行分析发现,猪DAI基因编码蛋白结构域和人、牛、鼠一样,其N端都具有两个DNA结合区。组织表达谱分析表明猪DAI mRNA主要表达于脾脏、肺脏、肾脏和小肠中。为了分析猪DAI是否能诱导I型干扰素的产生,采用荧光素酶报告系统检测发现在不同猪源细胞中超表达猪DAI均能在不同程度上激活转录因子IRF3(interferon regulatory factor 3)和NF-κB(nuclear factor-kappaB),并诱导IFN-p的产生,其DNA结合区和C端对激活IFN-p都是非常重要的。而通过RNA干扰技术把猪DAI沉默后则显著抑伟dsDNA和PRV诱导的IFN-β。这些结果提示DAI是猪天然免疫系统中的一个dsDNA的模式识别受体,在I型干扰素诱导的信号通路中具有重要作用。2.猪STING基因的克隆及其功能鉴定继DAI后,国际上三个研究小组几乎同时报道了另一个参与细胞内dsDNA诱导IFN-I表达的信号分子-STING(stimulator of interferon genes),但不同研究对STING的亚细胞定位及其在dsRNA和RNA病毒诱导IFN-I中的作用存在着分歧。根据与人STING基因序列有较高同源性的猪EST序列进行拼接并设计引物,从猪外周血单核细胞中扩增并获得猪STING全长cDNA。序列分析表明,猪STING开放阅读框全长为1137bp(GenBank收录号：FJ455509),编码378个氨基酸,含有1个内质网滞留序列。通过组织表达谱分析发现猪STING主要表达于脾脏、淋巴结和肺脏中。结构预测表明猪STING含有4个跨膜区,利用绿荧光蛋白做标记进行研究发现其主要定位于内质网,但也有部分定位于线粒体。超表达猪STING能有效激活转录因子IRF3和NF-κB,并诱导IFN-β的产生。利用RNAi下调猪STING的表达则抑制RNA病毒和DNA病毒在细胞中所诱导的IFN-β的表达。这些结果提示STING是猪天然免疫系统中的一个重要的调节因子,为进一步阐明STING在天然免疫中的作用提供了依据。3.PRV诱导β干扰素产生的分子机制研究伪狂犬病毒(PRV)属于疱疹病毒科,为双链DNA病毒。以前的研究表明,PRV感染能有效诱导机体天然免疫。但是迄今为止对于PRV是否诱导p干扰素(IFN-β)的产生及其分子机制还不清楚。采用IFN-β启动子荧光素酶报告系统检测发现PRV感染PK-15细胞(porcine kidney cell line)后能显著诱导IFN-β的产生,荧光定量RT-PCR检测也得到了相似的结果。为了阐明PRV感染PK-15细胞激活IFN-β的分子机制,运用RNAi技术和显性负调控突变体分别研究了胞内信号通路和TLR信号通路在PRV激活IFN-p中的作用。结果显示PRV可通过TLR(Toll-like receptor)的接头分子MyD88(myeloid differentiation factor-88)和胞内信号分子RIG-I(retinoic acid-inducible gene I)和VISA(virus-induced signaling adaptor)激活IFN-β。有趣的是,RNAi实验表明PRV感染PK-15细胞诱导IFN-β的表达需要IRF1(interferon regulatory factor 1)、IRF5(interferon regulatory factor 5)和IRF7(interferon regulatory factor 7)参与,却不需要IRF3。IRF3是干扰素调节因子(interferon regulatory factors, IRFs)家族中的重要转录因子之一,并且在很多病毒诱导的干扰素基因表达和抗病毒天然免疫反应中都具有重要作用。为了进一步验证IRFs与PRV感染激活IFN-β产生的关系,将各个干扰素调节因子的负调控突变体转染人胚胎肾细胞系(human embryonic kidney cell line, HEK293),然后用PRV感染,结果证明PRV感染HEK293细胞激活IFN-β产生也不需要转录因子IRF3的参与。这些结果提示PRV通过一种独特的机制调节天然免疫。但是,很多问题仍然未知,如PRV如何被信号分子识别以及具体的信号传递网络等。对这些问题的研究将有助于进一步了解DNA病毒诱导的天然免疫机制。4. TGEV诱导p干扰素产生的分子机制研究猪传染性胃肠炎病毒(TGEV)属于冠状病毒科冠状病毒属,为不分节段的单股正链RNA病毒。以前的研究表明,TGEV诱导IFN-I的表达。但是对其诱导IFN-I产生的机制并不清楚。我们利用荧光素酶报告系统和荧光定量检测发现TGEV在PK-15细胞中能显著激活IFN-β。进一步对TLR信号通路和胞内信号通路相关信号分子在TGEV诱导IFN-β中的作用的研究发现,TGEV可通过TLR信号通路的接头分子MyD88诱导IFN-β同时还依赖RIG-I、MDA5(melanoma differentiation-associated gene 5、STING、HMGB1(High mobility group box 1)、HMGB2(High mobility group box 2)、IRF5和IRF7等胞内信号分子,但与TRIF(TIR domain-containing adaptor inducing IFN-β)、IRF1及IRF3无关。对TGEV各结构蛋白诱导猪IFN-β启动子能力的研究发现,M蛋白(membrane protein)和N蛋白(nucleocapsid protein)能极显著地诱导IFN-β启动子活化,提示了其可能在TGEV诱导IFN-β中起着重要的作用。其具体的分子机理有待于进一步研究。更多还原

【Abstract】 The mechanism of viral infection and host immune response is one of the most important frontier research fields in virology. The host innate immune system senses invading viruses through specific molecular pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and then initiate an immune response. Upon recognition, the PRRs initiates a series of signaling, this leads to expression of a number of cytokines such as type I interferon (IFN-I). The secreted IFN-I bind to the receptors and initiate signaling that activates transcription of thousands of genes. The produced proteins collaborate to inhibit viral replication and spread. On the other hand, IFN-I activate innate immune cells to induce adaptive immune response, resulting in clearance of invading virus. Thus, IFN-I play a vital role in host antiviral response.Although much progress has been made in understanding the virus-triggered IFN-I signaling pathways in recent years, there are a lot of questions remaining to be elucidated. For example, the detailed mechanisms about how the newly signal proteins recognize virus and induce IFN-I production are not known yet. Also, are there unknown proteins involved in virus-triggered IFN-I signaling? How the immune system recognizes DNA viruses to induce the expression of type I interferon, and so on. In recent years, there have been growing interests in the swine immune system, because of its potential as a model for the study of the human immune system with a lower cost and high similarity. To reveal the molecular mechanism of IFN-I induction after virus infection in swine, we cloned and characterized two important signaling proteins from porcine cells, and studied the signal pathways in innate immune responses during PRV infection and TGEV infection, as the model of DNA viruses and RNA viruses, respectively. The main research works were as following:1. Molecular cloning and functional characterization of porcine DNA-dependent activator of IFN-regulatory factors (DAI)The DNA-dependent activator of IFN-regulatory factors (DAI) is the first identified cytosolic DNA sensor for intracellular DNA that triggers a signal for the production of type I interferon. Different observations indicate that, in addition to acting in a cell type-specific way, the function of DAI might also show inter-species differences. In order to investigate the role of porcine DAI (poDAI) in the type I interferon signaling pathway, based on the porcine genomic sequence obtained from the BLAST search, we cloned and sequenced a DAI cDNA isolated from porcine peripheral blood mononuclear cells (PBMC). The full-length cDNA of poDAI contains 1320 bp and encodes 439 amino acid residues (GenBank accession number FJ455510). Structural analysis with the SMART program indicated that poDAI contains two putative N-terminal DNA-binding domains. Similar DNA-binding domains have been identified in cattle, human and mouse DAI. poDAI mRNA expression was mainly detected in spleen, lung, kidney and small intestine. To investigate whether poDAI is involved in the type I interferon signaling pathway, porcine IFN-P promoter luciferase reporter system assays showed Over-expression of poDAI activated transcription factors IRF3 and NF-κB and induced IFN-P in different porcine cell lines, but to varying degrees. Deletion mutant analysis revealed that both the DNA-binding domains and the C-terminus are required for full activation of IFN-β. siRNA targeting poDAI significantly decreased poly(dAT:dAT)-or Pseudorabies virus(PRV)-induced IFN-P activation. These results indicate that DAI is an important immuno-regulator of the porcine innate immune system.2. Molecular cloning and functional characterization of porcine stimulator of interferon genes (STING)The human stimulator of interferon genes (STING) has been proposed by three independent research groups as an adaptor that functions in cytosolic DNA signal pathway. Although the three groups concluded that STING is an important signaling sensor in the cytosolic DNA pathway, they also came to some significantly different conclusions. To investigate the role of porcine STING (poSTING) in the type I interferon signaling pathway, based on the porcine EST sequence obtained from the BLAST search, we cloned and sequenced poSTING cDNA isolated from porcine peripheral blood mononuclear cells. The full-length cDNA of poSTING contains 1137 bp and encodes 378 amino acid residues (GenBank accession number FJ455509), contains one endoplasmic reticulum(ER) retention motif, RAR. poSTING mRNA expression was mainly detected in the spleen, lymph node and lung. Structural analysis with the SMART program indicated that poSTING contains four putative transmembrane(TM) domains at its N-terminus. poSTING was found to reside predominantly in the ER, and also in the mitochondrial membrane in PK-15 cells. Over-expression of poSTING activated both IRF3 and NF-κB to induce IFN-βproduction, while knockdown of poSTING significantly inhibited DNA virus and RNA virus induced IFN-P promoter activation and IFN-P mRNA production. Altogether, these results indicate that STING is an important regulator of porcine innate immune signaling. The results will help better understand the biological role(s) of STING in innate immunity during evolution.3. Studies on the molecular mechanism that PRV induced type I interferon productionPseudorabies Virus (PRV) is a swine alpha herpesvirus owning double-stranded DNA genome. Previous studies have demonstrated PRV infection triggered efficient immune response. However, the mechanism that PRV induced type I interferon production is still unclear. Using Real-time PCR and porcine IFN-βpromoter luciferase reporter system, we demonstrated that PRV infection in PK-15 cells up-regulated IFN-βgene transcription. To evaluate the detailed molecular mechanisms, we investigated the roles of TLR signal pathway and cytosolic signal pathway in IFN-P induction during PRV infection, by RNAi technique and dominant negative mutants. Our results showed that PRV-induced IFN-βwas related with the adaptor MyD88 in TLR signal pathway and cytosolic signal proteins RIG-I, and VISA. RNAi experiments showed that PRV-induced IFN-βin PK-15 cells require IRF1, IRF5, and IRF7. Interestingly, IRF3, the very important transcription factor for type I interferon production after virus infection, is nonessential for PRV-induced IFN-βin PK-15. Further study demonstrated that IRF3 is nonessential for PRV-induced IFN-βin HEK293 cells, either. Altogether, these results suggest that a distinct pathway utilized by PRV to regulate innate immunity. However, there are many questions remain unknown, such as how the host recognize PRV to trigger IFN-I signal pathway and how about the detailed signal transduction networks.4. Studies on the molecular mechanism that TGEV induced type I interferon productionTransmissible Gastroenteritis Virus (TGEV), a member of coronavirus species, is an enveloped RNA virus. Previous studies have demonstrated TGEV is able to induce IFN-I production. However, the mechanisms that TGEV induced type I interferon production are still unclear. Using Real-time PCR and porcine IFN-βpromoter luciferase reporter system, we demonstrated that TGEV infection in PK-15 cells up-regulated IFN-βgene transcription. To evaluate the mechanisms behind this, we investigated IFN-βinduction in TLR signal pathway and cytosolic signal pathway during TGEV infection. Our results demonstrated that TGEV induced IFN-βwas related with the adaptor MyD88 in TLR signal pathway, as well as several cytosolic signal proteins, such as RIG-I, MDA5, STING, HMGB1, HMGB2, IRF5, and IRF7. Whereas unrelated with TRIF, IRF1, and IRF3. Further study demonstrated that TGEV membrance(M) protein and encleocapsid(N) protein can activate IFN-βpromoter, indicating that this two proteins may involve in TGEV induced IFN-P production.更多还原