狂犬病病毒磷蛋白调控狂犬病病毒复制的分子机制研究

发布时间：2018-01-18 16:15

  本文关键词：狂犬病病毒磷蛋白调控狂犬病病毒复制的分子机制研究　出处：《浙江大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 狂犬病病毒 磷蛋白 细胞周期蛋白37 热休克蛋白90 稳定性 依赖和非依赖Hsp90结合的途径 病毒感染

【摘要】：狂犬病病毒(Rabies virus, RABV)隶属于弹状病毒科(Rhabdovidae family)基因1型狂犬病病毒属成员病毒(Lyssavirus),是人和动物狂犬病的病原体。如今世界范围内狂犬病病毒属的成员主要有七个基因型：基因1型(狂犬病病毒,RABV),基因2型(Lagos蝙蝠病毒,LBV),基因3型(Mokola病毒,MOKV),基因4型(Duvenhage病毒,DUVV),基因5型(欧洲蝙蝠1型狂犬病病毒,EBLV-1),基因6型(欧洲蝙蝠2型狂犬病病毒,EBLV-2),基因7型(澳大利亚蝙蝠狂犬病病毒,ABLV)。其中基因1型狂犬病病毒RABV对人和动物带来的危害最严重。其病毒结构蛋白磷蛋白(P)作为一种多功能的非蛋白激酶蛋白,能够参与病毒的转录与复制,也是狂犬病病毒的一种干扰素拮抗因子。迄今为止,虽有诸多研究表明P蛋白既能与RABV本身的病毒结构蛋白又能与宿主细胞蛋白互作并参与RABV的感染,但这方面的数据还是相当有限,该病毒的致病机制还远未明了。本研究阐明了细胞周期蛋白37 (Cdc37)依赖和非依赖热休克蛋白90 (Hsp90)结合的途径稳定P蛋白促进RABV感染的分子机制,旨在为将来抗RABV新药物靶标的研究开发提供可供参考的基础数据。1.P蛋白作为Hsp90客户蛋白的发现前期研究发现RABV感染过程中Hsp90能够被募集至病毒转录与复制场所内基氏小体(Negri bodies, NBs)中,提示Hsp90可能涉及RABV的感染。因此本研究以鼠神经瘤母细胞N2a为RABV感染模型,以基因1型疫苗毒株HEP-Flury为主要实验毒株,首先采用Hsp90特异性抑制剂分析其对RABV感染的影响。Hsp90抑制剂格尔德霉素(GA)及其衍生物烯丙基氨基格尔德霉素(17-AAG)的处理能够降低细胞内RABV病毒蛋白的累积水平、病毒的复制水平、病毒各基因的转录水平以及病毒的滴度。Hsp90抑制剂17-AAG的处理并不能影响蛋白合成抑制剂放线菌酮(CHX)对RABV N蛋白基因mRNA水平的抑制作用。由此提示,Hsp90影响RABV感染可能是通过影响病毒蛋白稳定性引发,而不是由其直接影响病毒基因的转录或mRNA的稳定性导致。然而Hsp90通过何种途径影响病毒蛋白稳定性?是否是通过蛋白酶体途径和自噬途径?为了验证这个假设,本研究通过免疫印迹实验发现,蛋白酶体抑制剂MG-132的处理未能抑制17-AAG对RABV病毒蛋白N和P的降解作用,而自噬阻断剂wortmannin的处理可阻断17-AAG对RABV病毒蛋白N和P的降解作用。由此提示,Hsp90抑制剂降解RABV病毒蛋白N和P可能是通过自噬途径进行。为了确证Hsp90抑制剂降解RABV病毒蛋白的自噬途径以及避免wortmannin药物的脱靶效应,借助RNAi敲降试验证明自噬途径关键成分LC3B的敲降同样可阻断17-AAG对RABV病毒蛋白N和P的降解作用。由此表明Hsp90抑制剂可引起RABV病毒蛋白N和/或P的自噬途径降解。由此同时,本研究构建了pSG5-N和pSG5-P两个携带SV40启动子的真核表达载体,证明了17-AAG能够特异降解真核表达的P蛋白,而对真核表达N蛋白没有影响。进一步的免疫共沉淀实验(Co-IP)结果表明,RABV天然性或外源性表达的P蛋白可与内源性或外源性表达的Hsp90互作。此外,Co-IP实验结果又发现狂犬病病毒属成员CVS-11、ABLV和MOKV三个毒株的P蛋白均能够与Hsp90发生结合。Hsp90与P蛋白的互作是所有狂犬病病毒属成员毒株共有的特性。由此表明,狂犬病病毒属成员毒株P蛋白是Hsp90的客户蛋白。2.Cdc37参与Hsp90-P复合物形成的发现Hsp90对其客户蛋白伴侣功能的发挥需要一大批辅助伴侣蛋白的支持。为了找寻参与Hsp90-P复合物形成的关键辅助伴侣蛋白,我们首先分析了Hsp90变构体抑制剂Celastrol对RABV病毒蛋白累积的影响。免疫印迹实验结果表明,Celastrol药物具有类似17-AAG药物的作用也能够降低RABV P蛋白累积水平。由此提示辅助伴侣蛋白Cdc37可能参与了Hsp90-P复合物的形成。接着共聚焦实验显示,RABV感染情况下含有P蛋白的NBs能够大量募集Cdc37; Co-IP实验结果进一步显示Hsp90-Cdc37-P三元复合物的形成。由此表明,P蛋白是Hsp90/Cdc37伴侣系统的客户蛋白。3.Cdc37募集P蛋白至Hsp90系统依赖和非依赖Hsp90结合途径的发现已有研究报道Cdc37募集蛋白激酶至Hsp90系统存在依赖和非依赖Hsp90结合的途径。而Cdc37对其他种类客户蛋白的募集途径不甚明了。那作为非蛋白激酶的RABVP蛋白,Cdc37对其募集至Hsp90系统是否也存在这两条途径或其他途径?在本研究中我们证明Cdc37参与Hsp90-P复合物形成的四条途径：缺失Hsp90结合区域的Cdc37截短体未能提升P蛋白的累积水平,而野生型Cdc37和Cdc37截短体Cdc37(aa 1-323)能够通过依赖Hsp90结合的途径募集P蛋白至Hsp90系统进而促进P蛋白的累积；破坏了与Hsp90结合能力的Cdc37M165和L206单点和双点突变体依然保持着与P蛋白的互作,并提升P蛋白的累积水平,而且能够模拟野生型Cdc37增强Hsp90-P之间的互作,由此指示野生型Cdc37还能够以一种非依赖Hsp90结合的方式募集P蛋白至Hsp90系统来保护P蛋白的稳定性：Cdc37的激活需要其Ser13位点的磷酸化修饰,非激活的Cdc37(将Cdc37 Ser13位点突变成Ala)并未减弱其与Hsp90和P蛋白的互作,而且能够募集P蛋白至Hsp90系统进而促进P蛋白的累积,由此指示非激活的Cdc37存在类似于野生型Cdc37依赖Hsp90结合的方式募集P蛋白至Hsp90系统进而对P蛋白进行稳定；破坏了与Hsp90结合能力的非激活Cdc37 M165和L206的单点和双点突变体也依然保持着与P蛋白的互作,并提升P蛋白的累积水平,而且能够模拟野生型Cdc37增强Hsp90-P之间的互作,由此指示非激活的Cdc37也能够以一种非依赖Hsp90结合的方式募集P蛋白至Hsp90系统来保护P蛋白的稳定性。4. Cdc37、Hsp90稳定P蛋白正调控RABV感染的发现首先免疫印迹实验结果显示RABV的感染能够诱导Cdc37和Hsp90蛋白的表达,由此提示Cdc37和Hsp90在RABV感染过程中可能起着正向调控作用。接着,采用真核过表达的方式研究Cdc37和Hsp90对RABV感染的影响,结果发现,过表达Hsp90和Cdc37能够从蛋白水平引起RABV病毒P蛋白的累积。然后我们通过免疫印迹实验发现,过表达Cdc37和Hsp90能够阻断蛋白合成抑制剂CHX处理情况下的P蛋白降解,而对CHX处理情况下的N蛋白的降解无影响,由此表明Cdc37和Hsp90是从蛋白稳定性水平而不是从蛋白合成水平影响RABV病毒P蛋白的累积。此外,采用RNAi干扰的方法降低Cdc37或Hsp90蛋白的表达能够显著降低病毒蛋白表达和病毒RNA合成的水平,并减少细胞内病毒粒子的释放量,由此证实了Cdc37和Hsp90在RABV感染的正向调控作用。最后,综上数据,Cdc37和Hsp90是通过稳定P蛋白促进P蛋白的累积进而影响RABV的感染。
[Abstract]:Rabies virus (Rabies virus RABV) belonging to Rhabdoviridae (Rhabdovidae family) gene of rabies virus type 1 virus (Lyssavirus), is a member of human and animal rabies pathogen. Members of the now world rabies virus has seven main genotypes: genotype 1 (rabies virus, RABV). Genotype 2 (Lagos bat virus, LBV gene, type 3 (Mokola) virus, MOKV gene, type 4 (Duvenhage) virus, DUVV), genotype 5 (European bat rabies virus type 1, type 6 (EBLV-1) gene, European bat rabies virus type 2, type 7 (EBLV-2) gene, Australia bat rabies virus, ABLV). The harm of genotype 1 RABV of rabies virus on human and animal is the most serious. The virus structural protein phosphoprotein (P) as a function of non protein kinase protein, transcription and replication of the virus can participate in, but also a kind of rabies virus Interferon antagonist. So far, there are many studies showed that P protein can with virus structural protein RABV itself and protein interaction with host cells and participate in RABV infection, but this data is quite limited, the pathogenic mechanism of the virus is unknown. This study illustrates the cell cycle protein 37 (Cdc37) - dependent and non dependent on heat shock protein 90 (Hsp90) way to stabilize P protein binding promotes the molecular mechanism of RABV infection, to study the future development of new anti RABV drugs and provide reference protein data base for the.1.P found as Hsp90 client proteins previously found that RABV infection can be in the process of Hsp90 to raise the virus transcription and replication sites Negri bodies (Negri bodies, NBs), suggesting that Hsp90 may be involved in RABV infection. This study in rat neuroblastoma cells N2a RABV infection model, on the basis of Because of the type 1 vaccine strain HEP-Flury as the main experimental strains, firstly using Hsp90 specific inhibitor analysis of its effect on RABV infection of the.Hsp90 inhibitor geldanamycin (GA) and its derivatives allyl amino geldanamycin (17-AAG) treatment can reduce the accumulation level of RABV virus protein in cells, virus replication level, the titer of 17-AAG.Hsp90 inhibitors of virus gene transcription and virus treatment did not affect the protein synthesis inhibitor cycloheximide (CHX) inhibition of RABV N protein gene mRNA levels. The results suggest that Hsp90 infection may be caused by influencing the effect of RABV virus protein stability, rather than by its direct impact on the stability caused by viral gene transcription or mRNA. However, the mechanism through which Hsp90 affect viral protein stability? Whether it is through the proteasome pathway and the autophagy pathway to confirm this? Assume that this research by Western blotting experiments showed that the degradation effect of proteasome inhibitor MG-132 treatment failed to inhibit 17-AAG protein of RABV virus N and P, and autophagy inhibitor treatment agent wortmannin can block the degradation effect of 17-AAG on RABV virus protein N and P. The results suggest that the inhibitor of Hsp90 degradation of RABV viral protein N and P is through the autophagy pathway. In order to confirm the autophagy pathway of Hsp90 inhibitors of RABV viral protein degradation and to avoid off target effects of wortmannin drugs, with the help of RNAi knockdown test proves that the key component of LC3B knockdown of autophagy pathway can also block the degradation effect of 17-AAG on RABV virus protein N and P. The result shows that the Hsp90 inhibitors can induce the autophagy pathway for degradation of RABV the virus protein N and / or P. At the same time, this study constructed the eukaryotic expression vector pSG5-N and pSG5-P two with SV40 promoter, and prove that 17-AAG can Enough specific degradation of eukaryotic expression of P protein, the eukaryotic expression did not affect N protein. Further co immunoprecipitation (Co-IP) results show that RABV natural or exogenous expression of P protein can interact with endogenous or exogenous expression of Hsp90 and Co-IP. In addition, the experimental results found that rabies virus the members of CVS-11, ABLV and three strains of MOKV P protein can occur with the combination of.Hsp90 and P protein interactions are all members of the genus strains of rabies virus common characteristics with Hsp90. This showed that rabies virus strain P protein members is a client of Hsp90 protein.2.Cdc37 involved in Hsp90-P formation of complexes found that Hsp90 play on the customers need a large number of chaperone function auxiliary chaperone support. In order to find in the formation of the Hsp90-P complex key auxiliary chaperone protein, we first analyzed the Hsp90 mutant inhibitor Celastro Effect of L on RABV virus protein accumulation. Western blot results showed that Celastrol drugs with similar 17-AAG drugs can reduce RABV P protein accumulation level. It suggests that the formation of auxiliary chaperone protein Cdc37 may be involved in the Hsp90-P complex. Then confocal experiment showed that P protein containing RABV infection by NBs to raise Cdc37 Co-IP; experimental results show the formation of three yuan compound Hsp90-Cdc37-P. It showed that the P protein is Hsp90/Cdc37 protein chaperone system customer recruitment of.3.Cdc37 P protein to Hsp90 dependent and independent pathways that Hsp90 binding has been reported to Hsp90 protein kinase Cdc37 recruitment system dependent and non dependent Hsp90 binding way. Cdc37 of other kinds of ways to raise the customer is not very clear. The protein as a non protein kinase RABVP protein, Cdc37 of its equity To set whether the Hsp90 system there are two ways or other way? In this study, we show that four ways Cdc37 is involved in the formation of the Hsp90-P complex: the cumulative loss of Hsp90 level with Cdc37 truncated to the promotion of the regional P protein, while the wild type Cdc37 and Cdc37 Cdc37 (AA 1-323) truncated to approach through the Hsp90 dependent recruitment of P proteins to the Hsp90 system and then promote the accumulation of P protein; damage remains the interaction with the P protein and Hsp90 binding ability of Cdc37M165 and L206 single and double point mutants, and increase the accumulation level of P protein, and can simulate the wild type Cdc37 enhanced interaction between Hsp90-P. This indicates the stability of wild type Cdc37 also can be a kind of independent way to raise P protein Hsp90 binding to Hsp90 system to protect P protein: activation of phosphorylation sites of Cdc37 Ser13 to the non. The activation of Cdc37 (Cdc37 Ser13 loci mutated into Ala) did not weaken its interaction with Hsp90 and P protein, and P protein could be raised to Hsp90 system and promote the accumulation of P protein, thus indicating non activated Cdc37 are similar to the wild-type Cdc37 dependent Hsp90 binding to raise P protein to Hsp90 system and the stability of P protein; destroy the binding ability with Hsp90 non single point M165 and activation of Cdc37 L206 and double point mutants also remained the interaction with the P protein, and increase the accumulation level of P protein, and can simulate the wild type Cdc37 enhanced interaction between Hsp90-P, thus indicating non activated Cdc37 to a non Hsp90 dependent way with the recruitment of P proteins to the Hsp90 system to protect the stability of.4. Cdc37 P protein, Hsp90 P protein stability regulation of RABV infection found that the first Western blot showed RABV The expression of Cdc37 and Hsp90 protein can induce infection, suggesting that Cdc37 and Hsp90 may play a positive role in the process of RABV infection. Then the eukaryotic expression of Cdc37 and Hsp90 on the impact of RABV infection results showed that over expression of Hsp90 and Cdc37 can induce the accumulation of RABV virus P protein from protein the level by Western blotting. Then we found that over expression of Cdc37 and Hsp90 could inhibit the P protein degradation of the protein synthesis inhibitor CHX treatment under the condition of CHX treatment had no effect on degradation of the N protein, which indicated that Cdc37 and Hsp90 are from the protein stability level rather than from the protein synthesis level affects the accumulation of RABV virus P protein. In addition, using RNAi interference reduced the expression of Cdc37 or Hsp90 protein can significantly reduce the level of virus protein expression and viral RNA synthesis, and decrease the intracellular virus The release of particles confirms the positive regulation role of Cdc37 and Hsp90 in RABV infection. Finally, in conclusion, Cdc37 and Hsp90 promote the accumulation of P protein by stabilizing P protein, thereby affecting RABV infection.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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