【作者】 王荡；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2011， 博士

【摘要】 天然免疫不仅是机体抵抗病原体(包括病毒、细菌等)侵袭的第一道防线,而且是激活获得性免疫的基础。作为一种重要的抗病毒因子,干扰素在天然免疫研究领域具有十分重要的地位。而在进化过程中,很多病毒具有了逃逸或抑制干扰素的能力。深入研究病毒如何调控干扰素产生是当前病毒学和免疫学研究热点。目前,对人和小鼠天然免疫系统已经研究的比较清楚,而对猪天然免疫系统的了解还知之甚少。主要体现在大量的猪源天然免疫信号分子和效应分子没有被克隆和鉴定,以及效应分子启动子荧光素酶报告系统等高通量检测信号通路的工具缺乏,这在一定程度上阻碍了病毒与猪天然免疫系统相互作用的深入研究。鉴于此,本课题以猪天然免疫信号通路为切入点,克隆了16个重要的猪天然免疫信号通路相关分子,并构建了猪天然免疫信号通路效应分子启动子荧光素酶报告系统,初步建立了研究猪天然免疫信号通路的技术平台。在此基础上,进一步系统分析了口蹄疫病毒非结构蛋白Lpro和3Cpro抑制干扰素产生的作用机制和分子细节。具体内容如下：1.猪IFN-β启动子及其NF-κB结合位点荧光素酶报告系统的建立为了建立检测猪β干扰素信号传导蛋白和基因的方法,在详细分析了猪IFN-β基因启动子区域后,采用PCR方法从猪基因组DNA中克隆了IFN-β基因的启动子片段,分别构建了含有猪IFN-p基因启动子的荧光素酶报告载体(pIFN-β-Luc)及含有4个重复的NF-κB结合位点序列的荧光素酶报告载体(4×NF-κB-Luc)。将荧光素酶报告载体转染PK-15细胞,在poly(I:C)或poly(dA:dT)的刺激下,荧光素酶的表达显著增加,说明荧光素酶报告载体构建正确。本研究为进一步开展猪β干扰素信号转导通路的研究奠定了基础。2.猪RANTES启动子荧光素酶报告系统的建立正常T细胞表达和分泌的活性调节蛋白(regulated upon activation, normal T-cells expressed and secreted, RANTES)是一种重要的趋化因子,在炎症以及免疫反应中发挥着重要作用。通过对本实验室克隆获得的猪RANTES基因启动子区域的分析,确定了其特征性激活转录元件的结合位点,并构建了包含启动子的一系列5’端缺失突变体的荧光素酶报告质粒,转染细胞发现猪RANTES基因启动子-220/+46的266bp核苷酸区域可以满足其在PK-15细胞中的基础转录活性。进一步研究发现,poly(I:C)或poly(dA:dT)能显著诱导猪RANTES启动子活性以及mRNA水平的表达,且呈时间和剂量依赖性。启动子缺失和突变实验发现,干扰素刺激应答元件对poly(I:C)或poly(dA:dT)诱导RANTES转录是必需的。此外,干扰素调节因子IRF-3和IRF-7在poly(I:C)或poly(dA:dT)诱导RANTES途径中也发挥了重要作用。3.猪p-干扰素启动子刺激物1 (IPS-1)等天然免疫信号通路基因的克隆RIG-I/MDA5是胞内识别dsRNA的重要模式识别受体,p干扰素启动子刺激物1 (IFN-βpromoter stimulator 1, IPS-1)(也称为MAVS/VISA/Cardif)作为RIG-I/MDA5的重要接头分子参与诱导Ⅰ型干扰素的产生。本研究运用RT-PCR和RACE-PCR从猪外周血单核细胞中克隆得到猪IPS-1的cDNA,序列分析发现,猪IPS-1基因开放读码框全长1575 bp,编码524个氨基酸；其编码蛋白N端为CARD区,中间是脯氨酸富集区,C端为跨膜区。该蛋白与小鼠、大鼠、猴、人和牛RIG-I相似物的同源性介于59-79%。半定量PCR显示猪IPS-1基因在多个不同的组织中均有表达,利用绿荧光蛋白作标记证实猪IPS-1主要定位于线粒体,且其C端的跨膜结构域对其线粒体定位是必需的。超表达猪IPS-1能显著激活转录因子IRF-3和NF-κB,并诱导IFN-β的产生。利用缺失突变体进一步分析发现IPS-1的CARD区和跨膜区在IPS-1诱导Ⅰ型干扰素的信号通路中都是必需的。此外,猪IPS-1的干扰分子能负调控poly(I:C)诱导IFN-β的能力,表明IPS-1是猪天然免疫信号通路中的一个重要调节因子。该研究为今后进一步探讨IPS-1在猪感染性疾病中的作用奠定了基础。除了猪IPS-1基因外,本研究还相继克隆得到RIG-I、MDA5、TRAF6等15个猪天然免疫信号通路中的重要分子。这为今后研究猪天然免疫奠定基础,同时也为研究猪病原与宿主相互作用提供便利工具。4. FMDV Lpro通过下调干扰素调节因子3/7的表达抑制dsRNA诱导的Ⅰ型干扰素的转录已有研究证实口蹄疫病毒前导蛋白(FMDV Lpro)能通过抑制核转录因子NF-κB的活性负调控IFN-β的产生。本研究利用荧光素酶报告系统和荧光定量PCR证实Lpro也通过下调干扰素调节因子IRF-3/7的表达抑制dsRNA诱导的IFN-αl/β的表达。此外,超表达Lpro能显著降低包括2’,5’-OAS、ISG54、IP-10和RANTES在内的多个IRF依赖的干扰素诱导基因的表达。Lpro突变体实验表明,Lpro抑制dsRNA诱导的IFN-αl/β启动子的激活以及降低IRF-3/7表达的能力不依赖于其切割eIF-4G的活性。这些结果说明FMDV能通过多种策略来抵抗宿主对病毒感染的应答。5. FMDV Lpro作为病毒编码的去泛素化酶负调控Ⅰ型干扰素产生本研究发现FMDV Lpro具有去泛素化蛋白酶活性。序列比对和生物信息学结构分析发现,Lpro第51位半胱氨酸(C51)和第148位组氨酸(H148)的催化活性位点在7个血清型的FMDV中均高度保守,并且其拓扑结构与已知的细胞内去泛素化酶USP14以及病毒编码的去泛素化酶SARS-CoV木瓜样蛋白酶(PLpro)高度同源。经纯化的或者体内表达的Lpro均可以将K48和K63连接形式的泛素从泛素化蛋白上去除。进一步实验证实,Lpro可以显著抑制RIG-I、TBK1、TRAF3和TRAF6的泛素化,这些分子的泛素化均参与Ⅰ型干扰素的激活。突变体实验发现,Lpro的催化活性以及SAP结构域对其去泛素化活性以及抑制Ⅰ型干扰素都是必需的。这些结果表明FMDV Lpro作为病毒编码的新型去泛素化蛋白酶参与抑制Ⅰ型干扰素的产生,揭示了FMDV抵抗宿主的抗病毒天然免疫反应的一种新的机制。6. FMDV Lpro通过抑制λ1干扰素的转录负调控λ1干扰素的抗病毒作用IFN-λ1是最近发现的一种新的Ⅱ型干扰素,具有广谱的抗病毒活性。本研究发现,经纯化的重组猪IFN-λ1处理IBRS-2细胞后,能显著的减少FMDV在IBRS-2细胞上的复制,且呈剂量依赖性。通过双荧光素酶以及荧光定量RT-PCR分析发现FMDV感染不能诱导IFN-λ1的产生,提示抑制IFN-λ1的产生是FMDV的另一种有效的免疫逃避策略。进一步研究发现FMDV Lpro抑制由poly(I:C)诱导的IFN-λ1启动子活性,说明FMDV Lpro参与调节IFN-λ1的表达。突变体实验表明Lpro催化活性以及SAP区域对其抑制poly(I:C)诱导IFN-λ1产生是必需的。这些结果表明IFN-λ1能抑制FMDV,但FMDV通过编码Lpro拮抗IFN-λ1的抗病毒作用。7. FMDV Lpro抑制dsRNA诱导的RANTES转录的分子机制研究趋化因子RANTES在炎症以及免疫反应中发挥着重要的作用。已有研究报道,与缺失Lpro的口蹄疫基因工程病毒相比,野生型FMDV能显著抑制RANTES的表达,但具体机制尚不清楚。本研究发现超表达FMDV Lpro能显著抑制poly(I:C)诱导的猪RANTES启动子活性以及mRNA水平的表达。此外,FMDV Lpro也能抑制IRF-3/7介导的RANTES启动子的激活。RANTES启动子的突变体实验发现Lpro是通过IRSE抑制poly(I:C)诱导的RANTES启动子的激活。Lpro突变体实验发现Lpro催化活性以及SAP区域对抑制poly(I:C)诱导RANTES产生是必需的。这一结果说明FMDV可能通过抑制RANTES调控宿主的炎症以及免疫反应。8. FMDV 3Cpro蛋白酶抑制Ⅰ、Ⅲ型干扰素产生的分子机制初步研究口蹄疫病毒3C蛋白(FMDV 3Cpro)是FMDV编码的重要蛋白酶之一,本研究发现3Cpro参与抑制Ⅰ型干扰素的产生,这说明FMDV通过编码多种蛋白来抵抗宿主对病毒感染的应答。进一步研究发现,3Cpro通过阻碍IRF-3/7的活化抑制IFN-α1/β启动子的激活。此外,超表达3Cpro能显著降低包括2’,5’-OAS、ISG54、IP-10、RANTES和IFN-λ1在内的多个IRF依赖的细胞因子的表达。总之,本研究初步建立了猪天然免疫信号通路研究的技术平台,利用该平台较为系统地分析口蹄疫病毒Lpro和3Cpro抑制干扰素和RANTES产生的作用机制和分子细节,揭示了口蹄疫病毒的免疫抑制特性和免疫逃逸策略,为今后开发更安全有效的新型口蹄疫疫苗奠定了理论基础。更多还原

【Abstract】 Innate immunity is not only the first defensive line to pathogen (including viruses and bacteria etc.) but also the basis to activate acquired immunity. Act as an important anti-virus factor, interferon plays a fundamental role in innate immunity research. And many viruses have the capability to escape or inhibit the effect of interferon in their evolution. It is a hot point in present virological and immunological research to deeply study for virus how to modulate the production of interferon.At present, the innate immunity of human and mice is relatively clear in researches, while little is known about the porcine innate immunity. A large amount of porcine signal molecules and effective molecules in innate immunity haven’t been cloned and identified, and the lack of luciferase reporting system for effective molecule promoter partly impedes deeply study at the interaction between virus and porcine innate immunity. Since this project took the signal pathway of porcine innate immunity as entry point to clone 16 important molecules related to signal pathway in porcine innate immunity and constructed Iuciferase reporting system for effective molecule promoter in porcine innate immunity, preliminarily build up technological platform to study signal pathway of porcine innate immunity. On the basis, to systematically analyze the mechanism and molecular details in the production of interferon inhibited by non-structure protein Lpro and 3Cpro in FMDV. The main studies were as following:1. Construction and identification of luciferase reporter gene vectors directed by porcine IFN-βpromoter and its NF-kB binding siteTo establish a method for detection of porcine proteins and genes related to IFN-βsignal transduction, we analyzed the regulatory elements that regulate the transcrip tion of porcine IFN-βgene. The promoter region of porcine IFN-βgene and its four copies NF-kB (nuclear factor kB) binding site regions were amplified from porcine genomic DNA by PCR and were cloned into promoter-free plasmid pGL3-basic. Then these constructs were transiently transfected into PK-15 cells and luciferase activities were measured with or without the transfection of poly(I:C) or poly(dA:dT). Higher expression of luciferase was obviously detected in PK-15 cells transfected with poly(I:C) or poly(dA:dT). These reporter constructs are important tools for investigation of porcine IFN-βsignaling transduction pathways.2. Molecular cloning of the porcine RANTES promoter:functional characterization of dsDNA/dsRNA response elements in PK-15 cellsThe chemokine RANTES (regulated upon activation, normal T-cells expressed and secreted) plays an essential role in inflammation and immune response. In this study, we cloned the nucleotide sequence of the 5’-flanking region of the porcine RANTES (poRANTES) gene and characterized the regulatory elements that activate transcription. Analyses of a series of 5’deletion constructs demonstrated that a 266 bp region (-220/+46) that spanned the potential transcription start site of the poRANTES gene was sufficient to activate transcription in PK-15 cells. Furthermore, our results indicated that dsDNA/dsRNA significantly induced poRANTES promoter activity and expression of mRNA levels in a time-and dose-dependent manner. Promoter deletions and mutagenesis experiments indicated that an interferon-stimulated responsive element (ISRE) was critical for dsDNA/dsRNA-induced poRANTES transcription. In addition, porcine interferon regulatory factor 3 (IRF-3) and IRF-7 play important roles in dsDNA/dsRNA-induced poRANTES expression.3. Molecular cloning and functional characterization of porcine IFN-βpromoter stimulator 1 (IPS-1)The IFN-βpromoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that senses intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, the porcine IPS-1 cDNA was cloned using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR from porcine peripheral blood monouclear cells. The open reading frame of porcine IPS-1 consists of 1575 bp encoding 524 amino acids. The putative porcine IPS-1 protein contains an N-terminal CARD-like domain, a proline-rich domain in the middle, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts ranging from 59 to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 could produce type I IFN, and activate IRF-3/7 (interferon regulatory factor 3/7) and NF-kB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-βpromoter activation in PK-15 cells could be significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.Besides porcine IPS-1 gene, this research in succession cloned RIG-I、MDA5、TRAF6 etc 15 important molecules in signal pathway of porcine innate immunity. It laid basis on the research in porcine innate immunity in future and provided convenient tool to study the interaction between porcine pathogen and host.4. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levelsThe leader proteinase (Lpro) of FMDV has been identified as an interferon-β(IFN-β) antagonist that disrupts the integrity of transcription factor nuclear factor kB (NF-kB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-αl/βexpression caused by Lpro was also associated with a decrease of IRF-3/7 in protein levels, two critical transcription factors for activation of IFN-a/β. Furthermore, overexpression of Lpro significantly reduced the transcription ofro multiple IRF-responsive genes including 2’,5’-OAS, ISG54, IP-10, and RANTES. Screening Lpro mutants indicated that the ability to process eIF-4G of Lpro is not required for suppressing dsRNA-induced activation of the IFN-al/βpromoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-kB, Lpro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.5. The leader proteinase of foot-and-mouth disease virus negatively regulates type I interferon pathway by acting as a viral deubiquitinaseHere, we demonstrate that FMDV Lpro has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His 148) are highly conserved in FMDV Lpro of all seven serotypes and the topology of FMDV Lpro is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral deubiquitylation enzyme (DUB). Both purified Lpro protein and in vivo ectopically expressed Lpro removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48-and lysine-63-linked polyubiquitin chains. Furthermore, Lpro significantly inhibited ubiquitination of RIG-I TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6) and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lpro that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lpro as well as its ability to block signaling to the IFN-βpromoter. Collectively, these results demonstrate that FMDV Lpro possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.6. FMDV could be inhibited by interferon-Iambda 1 and had mechanisms to block this actionFMDV causes an economically important disease in swine-producing area, and interferon lambda 1 (IFN-λ1), a newly identified type III interferon, has antiviral activity against a broad spectrum of viruses. In this study, we found that replication of FMDV in IBRS-2 cells was significantly reduced following treatment with the purified recombinant porcine IFN-λ1 in a dose-dependent manner. However, FMDV could not activate IFN-λ1 promoter in IBRS-2 cells, and the activity of IFN-λ1 promoter was much lower than that triggered by poly(I:C). Furthermore, we found that the leader proteinase (Lpro) of FMDV is involved in IFN-λ1 regulation. The obtained results showed that FMDV Lpro inhibited poly(I:C)-induced IFN-λ1 promoter activity. Screening Lpro mutants indicated that the catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of Lpro were required for suppressing dsRNA-induced IFN-λ1 production. In conclusion, our results suggested that FMDV could be inhibited by IFN-λ1 and had mechanisms to inhibit this action.7. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced RANTES transcription in PK-15 cellsThe chemokine RANTES plays an essential role in inflammation and immune response. Previous study has been demonstrated that infection with wild-type (WT) foot-and-mouth disease virus inhibits the expression of RANTES compared to infection with a genetically engineered mutant lacking the leader protein (Lpro) coding region in PK-15 cells. However, a complete analysis of the promoter cis-regulatory elements and nuclear factors involved in this inhibition of RANTES gene transcription has not been fully elucidated. In this study, we showed that transfection of PK-15 cells with Lpro of FMDV expression plasmid, in the absence of other FMDV proteins, inhibited dsRNA-induced RANTES in luciferase activity and mRNA transcription. Promoter mutagenesis experiments indicate that interferon-stimulated responsive element (ISRE) was important for the ability of Lpro to inhibit dsRNA-induced the RANTES promoter. Furthermore, the overexpression of Lpro also inhibited IRF-3/7-mediated activation of the RANTES promoter. Screening Lpr0 mutants indicated that the catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of Lpro were required for suppressing dsRNA-induced RANTES production. These findings reveal one of the important mechanisms underlying the innate immune evasion by FMDV during infection.8. Foot-and-mouth disease virus 3C proteinase inhibits type IIFN transcription The 3C proteinase (Lpro) of FMDV has proteinase activity and is involved in processing the viral polyprotein. In this study, our results show that 3Cpro is involved in the inhibition of type I IFN response, suggesting multiple proteins used by FMDV to counteract the immune response to viral infection. FMDV 3Cpro negatively regulates IFN-al/βexpression by disrupting activation of IRF-3/7. Furthermore, overexpression of 3Cpro significantly reduced the transcription of multiple IRF-responsive genes including 2’,5’-OAS, ISG54, IP-10, RANTES, and IFN-λ1.In a word, this project preliminarily build up technological platform to study signal pathway of porcine innate immunity and systematically analyzed the mechanism and molecular details in the production of interferon inhibited by non-structure protein Lpro and 3Cpro in FMDV, proclaimed the immune inhibitory characteristic and immune escaping strategy, which established theoretical basis to exploit safer and more effective vaccine of FMD from now on.更多还原