副黏病毒Ⅴ蛋白拮抗MAVS介導(dǎo)的Ⅰ型干擾素通路的研究
發(fā)布時間：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屬(AvuLavirus)的禽副黏病毒Ⅰ型(APMV-1)。NDV基因編碼六種主要結(jié)構(gòu)蛋白組成并且研究較多的P基因可以通過RNA編碼產(chǎn)生兩個非結(jié)構(gòu)蛋白Ⅴ蛋白和W蛋白。維甲酸誘導(dǎo)基因-Ⅰ(retinoic acid induced gene-Ⅰ,RIG-Ⅰ)是識別病毒RNA激發(fā)Ⅰ型IFN產(chǎn)生的重要傳感器,線粒體抗病毒信號蛋白MAVS(Mitochondrial antiviral signaling protein,IPS-1/VISA/Cardif也是它的名稱)蛋白是RIG-Ⅰ樣受體在先天性抗病毒免疫中的接頭蛋白,新城疫病毒感染時可誘導(dǎo)宿主細胞產(chǎn)生免疫性干擾素(IFN)來抑制病毒增殖,文獻記載新城疫病毒編碼的Ⅴ蛋白等多種蛋白阻止IFN的抗病毒功能,因此病毒Ⅴ蛋白與細胞MAVS蛋白是否存在相互作用近年來成為新的研究熱點。NDV的Ⅴ蛋白結(jié)構(gòu)同同類的其他副黏病毒的Ⅴ蛋白具有相似的特殊結(jié)構(gòu),發(fā)現(xiàn)副黏病毒通過RNA編碼,產(chǎn)生Ⅴ蛋白具有阻斷IFN抗病毒活性的功能。已有研究發(fā)現(xiàn)NDV Ⅴ蛋白對MAVS信號通路有降解作用,本實驗通過外源轉(zhuǎn)染副黏病毒Ⅴ蛋白,檢測其對細胞MAVS蛋白表達及IFN-β生成量的影響,結(jié)果說明副黏病毒Ⅴ蛋白通過泛素-蛋白酶體途徑降解MAVS蛋白,并且病毒是通過MAVS蛋白水平干擾IFN的產(chǎn)生。實驗還通過獲得MAVS不同片段的克隆來研究病毒Ⅴ蛋白與MAVS的相互作用,發(fā)現(xiàn)副黏病毒Ⅴ蛋白與MAVS作用的特異性�？傊�,實驗研究驗證病毒Ⅴ蛋白的在拮抗干擾素中的重要作用。一、NDV誘導(dǎo)MAVS降解,但下游IFN信號通路仍然激活本研究使用了 NDV、SeV感染HeLa6、9、12、24h后檢測MAVS表達水平,發(fā)現(xiàn)在病毒感染24h降解MAVS。本實驗還證明了 NDV以一種劑量依賴效應(yīng)誘導(dǎo)MAVS降解,并且NDV還可以降解外源轉(zhuǎn)染的MAVS。為了驗證細胞感染NDV后MAVS下游信號通路的激活情況,本實驗在HeLa細胞感染 NDV3、6、9、12h 后 Western-Blot 檢測 TBK1/p-TBK1/pcbp2/Mavs/p-IRF-3 蛋白表達量,檢測結(jié)果顯示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顯。結(jié)果表明,影響干擾素產(chǎn)生的TBK1、IRF-3等的激活可能是由其他信號通路激活引起的,后續(xù)實驗有待進一步研究。二、NDV Ⅴ蛋白降解MAVS研究發(fā)現(xiàn)構(gòu)建NDV的Flag-V基因能夠降解細胞的MAVS蛋白,為進一步的驗證是否是NDV的Ⅴ蛋白在細胞先天性免疫中發(fā)揮MAVS降解的作用,實驗通過已構(gòu)建的Flag-Ⅴ蛋白轉(zhuǎn)染HeLa細胞和A549細胞,檢測MAVS表達水平。為了證明RIG-MDA5信號通路是否被NDV的Ⅴ蛋白干擾,通過熒光素酶實驗從干擾素啟動子水平檢測轉(zhuǎn)染不同濃度梯度的Flag-V和Flag-MAVS后對細胞IFN-β產(chǎn)生的影響,結(jié)果顯示隨著轉(zhuǎn)染Flag-V濃度的升高,在轉(zhuǎn)染Flag-MAVS時IFN-β產(chǎn)生水平逐漸降低,而轉(zhuǎn)染TLR3下游的接頭蛋白Trif則沒有影響。三、副黏病毒Ⅴ蛋白降解MAVS研究中我們構(gòu)建副黏病毒的SeV、MEV驗證隨著轉(zhuǎn)染Ⅴ濃度的升高,外源轉(zhuǎn)染MAVS及NDV感染誘導(dǎo)的IFN-β水平是降低。為了進一步論證NDV Ⅴ蛋白能否降解MAVS,本實驗通過實驗室已有的ZJ1病毒Ⅴ蛋白突變株以及WT感染HeLa細胞后觀察MAVS降解情況,結(jié)果證明病毒Ⅴ蛋白缺失后細胞MAVS蛋白未出現(xiàn)降解情況,也確證了 NDV Ⅴ蛋白對MAVS的作用。四、NDV通過泛素-蛋白酶體途徑降解MAVS通過293T細胞轉(zhuǎn)染HA-K48,24h后感染NDV后做CO-IP實驗,不感染作對照,觀察是否MAVS為泛素化降解,結(jié)果顯示轉(zhuǎn)染HA-K48后感染NDV的蛋白互作實驗說明是NDV通過蛋白酶體泛素化降解MAVS。MAVS蛋白降解通過兩條通路,一條是蛋白酶體途徑,另一條是通過自噬通路。為了驗證這兩個通路對NDV感染后對MAVS 降解的影響,本實驗通過用 MG132、CQ、E64d/PepstatinA、wortmannin 不同濃度處理檢測MAVS降解是否延遲,結(jié)果顯示自噬阻斷藥物處理HeLa細胞后并不影響MAVS的降解,而蛋白酶體抑制劑MG132則抑制了 MAVS的降解,說明其主要是通過泛素-蛋白酶體途徑降解。五、NDV Ⅴ蛋白與MAVS的特異性反應(yīng)有研究已證明PCBP2可以與MAVS180-540這一片段相互作用,由于本實驗已排除PCBP2降解MAVS的作用,為了論證病毒Ⅴ蛋白與MAVS分段之間作用的特異性,本實驗在293細胞共轉(zhuǎn)染NDV Ⅴ蛋白與MAVS分段,然后Western-Blot檢測相互作用的特異性反應(yīng)。結(jié)果顯示NDV Ⅴ蛋白與細胞MAVS蛋白的360-540片段發(fā)生特異性反應(yīng)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65
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