副黏病毒Ⅴ蛋白拮抗MAVS介导的Ⅰ型干扰素通路的研究

发布时间：2019-05-17 01:18

【摘要】：新城疫病毒(Newcastle Disease virus,NDV)是副黏病毒科禽腮腺炎病毒属(AvuLavirus)的禽副黏病毒Ⅰ型(APMV-1)。NDV基因编码六种主要结构蛋白组成并且研究较多的P基因可以通过RNA编码产生两个非结构蛋白Ⅴ蛋白和W蛋白。维甲酸诱导基因-Ⅰ(retinoic acid induced gene-Ⅰ,RIG-Ⅰ)是识别病毒RNA激发Ⅰ型IFN产生的重要传感器,线粒体抗病毒信号蛋白MAVS(Mitochondrial antiviral signaling protein,IPS-1/VISA/Cardif也是它的名称)蛋白是RIG-Ⅰ样受体在先天性抗病毒免疫中的接头蛋白,新城疫病毒感染时可诱导宿主细胞产生免疫性干扰素(IFN)来抑制病毒增殖,文献记载新城疫病毒编码的Ⅴ蛋白等多种蛋白阻止IFN的抗病毒功能,因此病毒Ⅴ蛋白与细胞MAVS蛋白是否存在相互作用近年来成为新的研究热点。NDV的Ⅴ蛋白结构同同类的其他副黏病毒的Ⅴ蛋白具有相似的特殊结构,发现副黏病毒通过RNA编码,产生Ⅴ蛋白具有阻断IFN抗病毒活性的功能。已有研究发现NDV Ⅴ蛋白对MAVS信号通路有降解作用,本实验通过外源转染副黏病毒Ⅴ蛋白,检测其对细胞MAVS蛋白表达及IFN-β生成量的影响,结果说明副黏病毒Ⅴ蛋白通过泛素-蛋白酶体途径降解MAVS蛋白,并且病毒是通过MAVS蛋白水平干扰IFN的产生。实验还通过获得MAVS不同片段的克隆来研究病毒Ⅴ蛋白与MAVS的相互作用,发现副黏病毒Ⅴ蛋白与MAVS作用的特异性。总之,实验研究验证病毒Ⅴ蛋白的在拮抗干扰素中的重要作用。一、NDV诱导MAVS降解,但下游IFN信号通路仍然激活本研究使用了 NDV、SeV感染HeLa6、9、12、24h后检测MAVS表达水平,发现在病毒感染24h降解MAVS。本实验还证明了 NDV以一种剂量依赖效应诱导MAVS降解,并且NDV还可以降解外源转染的MAVS。为了验证细胞感染NDV后MAVS下游信号通路的激活情况,本实验在HeLa细胞感染 NDV3、6、9、12h 后 Western-Blot 检测 TBK1/p-TBK1/pcbp2/Mavs/p-IRF-3 蛋白表达量,检测结果显示NDV感染前期不引起p-TBK1、p-IRF-3的激活,而在感染后期激活明显。结果表明,影响干扰素产生的TBK1、IRF-3等的激活可能是由其他信号通路激活引起的,后续实验有待进一步研究。二、NDV Ⅴ蛋白降解MAVS研究发现构建NDV的Flag-V基因能够降解细胞的MAVS蛋白,为进一步的验证是否是NDV的Ⅴ蛋白在细胞先天性免疫中发挥MAVS降解的作用,实验通过已构建的Flag-Ⅴ蛋白转染HeLa细胞和A549细胞,检测MAVS表达水平。为了证明RIG-MDA5信号通路是否被NDV的Ⅴ蛋白干扰,通过荧光素酶实验从干扰素启动子水平检测转染不同浓度梯度的Flag-V和Flag-MAVS后对细胞IFN-β产生的影响,结果显示随着转染Flag-V浓度的升高,在转染Flag-MAVS时IFN-β产生水平逐渐降低,而转染TLR3下游的接头蛋白Trif则没有影响。三、副黏病毒Ⅴ蛋白降解MAVS研究中我们构建副黏病毒的SeV、MEV验证随着转染Ⅴ浓度的升高,外源转染MAVS及NDV感染诱导的IFN-β水平是降低。为了进一步论证NDV Ⅴ蛋白能否降解MAVS,本实验通过实验室已有的ZJ1病毒Ⅴ蛋白突变株以及WT感染HeLa细胞后观察MAVS降解情况,结果证明病毒Ⅴ蛋白缺失后细胞MAVS蛋白未出现降解情况,也确证了 NDV Ⅴ蛋白对MAVS的作用。四、NDV通过泛素-蛋白酶体途径降解MAVS通过293T细胞转染HA-K48,24h后感染NDV后做CO-IP实验,不感染作对照,观察是否MAVS为泛素化降解,结果显示转染HA-K48后感染NDV的蛋白互作实验说明是NDV通过蛋白酶体泛素化降解MAVS。MAVS蛋白降解通过两条通路,一条是蛋白酶体途径,另一条是通过自噬通路。为了验证这两个通路对NDV感染后对MAVS 降解的影响,本实验通过用 MG132、CQ、E64d/PepstatinA、wortmannin 不同浓度处理检测MAVS降解是否延迟,结果显示自噬阻断药物处理HeLa细胞后并不影响MAVS的降解,而蛋白酶体抑制剂MG132则抑制了 MAVS的降解,说明其主要是通过泛素-蛋白酶体途径降解。五、NDV Ⅴ蛋白与MAVS的特异性反应有研究已证明PCBP2可以与MAVS180-540这一片段相互作用,由于本实验已排除PCBP2降解MAVS的作用,为了论证病毒Ⅴ蛋白与MAVS分段之间作用的特异性,本实验在293细胞共转染NDV Ⅴ蛋白与MAVS分段,然后Western-Blot检测相互作用的特异性反应。结果显示NDV Ⅴ蛋白与细胞MAVS蛋白的360-540片段发生特异性反应。
[Abstract]:Newcastle disease virus (NDV) is an avian paramyxovirus type I (APMV-1) of Paramyxoviridae. The NDV gene encodes six major structural proteins and studies more P genes to produce two non-structural protein V and W proteins by RNA coding. Retinoic acid-induced gene-I (RIG-I) is an important sensor for the identification of viral RNA-excited type I IFN, and the mitochondrial anti-viral signal protein (MVS) protein (IPS-1/ VISA/ Cardif is also its name) protein is the linker protein of the RIG-I-like receptor in the innate anti-viral immunity, in that event of a newcastle disease virus infection, the host cell can be induce to produce an immune interferon (IFN) to inhibit the proliferation of the virus, Therefore, whether the interaction of the virus V protein with the cell MAVS protein has become a new research hotspot in recent years. The V-protein structure of NDV has a similar special structure with the V-protein of the other paramyxovirus of the same type, and it is found that the paramyxovirus is encoded by RNA, and the V-protein has the function of blocking the anti-viral activity of the IFN. The effect of NDV V protein on the expression of MVS protein and the amount of IFN-I was detected by transfecting the paramyxovirus V. The results showed that the V-protein of the paramyxovirus degrades the MAVS protein through the ubiquitin-proteasome pathway. And the virus is the production of the IFN by the level of the MAVS protein. The interaction between the virus V protein and the MAVS was also studied by the cloning of the different fragments of the MAVS, and the specificity of the effect of the paramyxovirus V protein and the MVS was found. In conclusion, the experimental study verifies the important role of the virus V protein in antagonizing the interferon. One, NDV induced the degradation of MVS, but the downstream IFN signal pathway still activated this study to use NDV, SeV-infected HeLa6,9,12, and 24 hours to detect the level of MAVS expression, and it was found that the MAVS was degraded at 24 h of viral infection. The experiment also demonstrated that NDV can induce the degradation of MVS with a dose-dependent effect, and NDV can also degrade the foreign-transfected MVS. In order to verify the activation of the signal pathway after NDV3,6,9 and 12 h after the infection of NDV3,6,9 and 12 h, the expression of TBK1/ p-TBK1/ pcbp2/ Mavs/ p-IRF-3 was detected by Western-Blot after the infection of NDV3,6,9 and 12 h in HeLa cells. The results show that the activation of TBK1, IRF-3 and so on may be caused by other signal pathway activation, and the follow-up experiment is to be further studied. 2. The NDV-V protein degradation MAVS study found that the FLAG-V gene of NDV can degrade the MAVS protein of the cells, and to further verify whether the V-protein of NDV can play the role of the MVS degradation in the cell first immunity, the experiment is carried out through the constructed Flag-V protein to transfect the HeLa cells and the A549 cells, And the expression level of the MVS was detected. In order to prove whether the signal path of the RIG-MDA5 signal is interfered by the V-protein of NDV, the effect of the Flag-V and Flag-MAVS of different concentration gradients on the cell IFN-antigen is detected by the luciferase experiment from the interferon promoter level, and the results show that with the increase of the transfection Flag-V concentration, At the time of transfection of Flag-MAVS, the level of IFN-antigen production decreased gradually, while the linker protein Trif downstream of the transfection TLR3 had no effect. In the MVS study, we constructed the SeV and MEV of the paramyxovirus, and with the increase of the level of the transfectant V, the level of the IFN-V induced by the exogenous transfection of the MVS and NDV infection was reduced. In order to further demonstrate the ability of the NDV V protein to degrade the MAVS, this experiment observed the degradation of the MVS after the mutation of the ZJ1 virus V protein mutant and the WT infected HeLa cells in the laboratory, and the results showed that the cell MAVS protein did not degrade after the deletion of the virus V. The effect of NDV V protein on MVS was also confirmed. and 4, the NDV is degraded through the ubiquitin-proteasome pathway to degrade the MAVS through 293T cells to be transfected into the HA-K48, after 24 hours, the NDV is infected with NDV, and the CO-IP experiment is performed, and the control is not carried out, and whether the MAVS is ubiquitinated degradation is observed, The results showed that the interaction of the protein with NDV after transfection of HA-K48 was demonstrated by the proteasome ubiquitination of the MVS. The degradation of the MAVS protein through two pathways, one is the proteasome pathway, and the other through the autophagy pathway. In order to verify the effect of these two pathways on the degradation of MVS after NDV infection, the results showed that the degradation of MVS was not affected after the treatment of HeLa cells by autophagy. The proteasome inhibitor MG132 inhibits the degradation of the MAVS, indicating that it is primarily degraded by the ubiquitin-proteasome pathway. V. The specific reaction of NDV V and MVS has shown that PCB P2 can interact with the segment of MAVS180-540, and as this experiment has ruled out the effect of PCBP2 on the degradation of MAVS, in order to demonstrate the specificity of the action between the virus V protein and the MVS segment, In this experiment, the NDV-V protein was co-transfected with the MVS in 293 cells, and then the specific reaction of the interaction was detected by Western-Blot. The results showed that the NDV V protein was specific to the 360-540 fragment of the cell MAVS protein.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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