發(fā)布時間：2018-01-18 16:15

  本文關(guān)鍵詞：狂犬病病毒磷蛋白調(diào)控狂犬病病毒復(fù)制的分子機(jī)制研究　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 狂犬病病毒 磷蛋白 細(xì)胞周期蛋白37 熱休克蛋白90 穩(wěn)定性 依賴和非依賴Hsp90結(jié)合的途徑 病毒感染

【摘要】：狂犬病病毒(Rabies virus, RABV)隸屬于彈狀病毒科(Rhabdovidae family)基因1型狂犬病病毒屬成員病毒(Lyssavirus),是人和動物狂犬病的病原體。如今世界范圍內(nèi)狂犬病病毒屬的成員主要有七個基因型：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(歐洲蝙蝠1型狂犬病病毒,EBLV-1),基因6型(歐洲蝙蝠2型狂犬病病毒,EBLV-2),基因7型(澳大利亞蝙蝠狂犬病病毒,ABLV)。其中基因1型狂犬病病毒RABV對人和動物帶來的危害最嚴(yán)重。其病毒結(jié)構(gòu)蛋白磷蛋白(P)作為一種多功能的非蛋白激酶蛋白,能夠參與病毒的轉(zhuǎn)錄與復(fù)制,也是狂犬病病毒的一種干擾素拮抗因子。迄今為止,雖有諸多研究表明P蛋白既能與RABV本身的病毒結(jié)構(gòu)蛋白又能與宿主細(xì)胞蛋白互作并參與RABV的感染,但這方面的數(shù)據(jù)還是相當(dāng)有限,該病毒的致病機(jī)制還遠(yuǎn)未明了。本研究闡明了細(xì)胞周期蛋白37 (Cdc37)依賴和非依賴熱休克蛋白90 (Hsp90)結(jié)合的途徑穩(wěn)定P蛋白促進(jìn)RABV感染的分子機(jī)制,旨在為將來抗RABV新藥物靶標(biāo)的研究開發(fā)提供可供參考的基礎(chǔ)數(shù)據(jù)。1.P蛋白作為Hsp90客戶蛋白的發(fā)現(xiàn)前期研究發(fā)現(xiàn)RABV感染過程中Hsp90能夠被募集至病毒轉(zhuǎn)錄與復(fù)制場所內(nèi)基氏小體(Negri bodies, NBs)中,提示Hsp90可能涉及RABV的感染。因此本研究以鼠神經(jīng)瘤母細(xì)胞N2a為RABV感染模型,以基因1型疫苗毒株HEP-Flury為主要實(shí)驗(yàn)毒株,首先采用Hsp90特異性抑制劑分析其對RABV感染的影響。Hsp90抑制劑格爾德霉素(GA)及其衍生物烯丙基氨基格爾德霉素(17-AAG)的處理能夠降低細(xì)胞內(nèi)RABV病毒蛋白的累積水平、病毒的復(fù)制水平、病毒各基因的轉(zhuǎn)錄水平以及病毒的滴度。Hsp90抑制劑17-AAG的處理并不能影響蛋白合成抑制劑放線菌酮(CHX)對RABV N蛋白基因mRNA水平的抑制作用。由此提示,Hsp90影響RABV感染可能是通過影響病毒蛋白穩(wěn)定性引發(fā),而不是由其直接影響病毒基因的轉(zhuǎn)錄或mRNA的穩(wěn)定性導(dǎo)致。然而Hsp90通過何種途徑影響病毒蛋白穩(wěn)定性?是否是通過蛋白酶體途徑和自噬途徑?為了驗(yàn)證這個假設(shè),本研究通過免疫印跡實(shí)驗(yàn)發(fā)現(xiàn),蛋白酶體抑制劑MG-132的處理未能抑制17-AAG對RABV病毒蛋白N和P的降解作用,而自噬阻斷劑wortmannin的處理可阻斷17-AAG對RABV病毒蛋白N和P的降解作用。由此提示,Hsp90抑制劑降解RABV病毒蛋白N和P可能是通過自噬途徑進(jìn)行。為了確證Hsp90抑制劑降解RABV病毒蛋白的自噬途徑以及避免wortmannin藥物的脫靶效應(yīng),借助RNAi敲降試驗(yàn)證明自噬途徑關(guān)鍵成分LC3B的敲降同樣可阻斷17-AAG對RABV病毒蛋白N和P的降解作用。由此表明Hsp90抑制劑可引起RABV病毒蛋白N和/或P的自噬途徑降解。由此同時,本研究構(gòu)建了pSG5-N和pSG5-P兩個攜帶SV40啟動子的真核表達(dá)載體,證明了17-AAG能夠特異降解真核表達(dá)的P蛋白,而對真核表達(dá)N蛋白沒有影響。進(jìn)一步的免疫共沉淀實(shí)驗(yàn)(Co-IP)結(jié)果表明,RABV天然性或外源性表達(dá)的P蛋白可與內(nèi)源性或外源性表達(dá)的Hsp90互作。此外,Co-IP實(shí)驗(yàn)結(jié)果又發(fā)現(xiàn)狂犬病病毒屬成員CVS-11、ABLV和MOKV三個毒株的P蛋白均能夠與Hsp90發(fā)生結(jié)合。Hsp90與P蛋白的互作是所有狂犬病病毒屬成員毒株共有的特性。由此表明,狂犬病病毒屬成員毒株P(guān)蛋白是Hsp90的客戶蛋白。2.Cdc37參與Hsp90-P復(fù)合物形成的發(fā)現(xiàn)Hsp90對其客戶蛋白伴侶功能的發(fā)揮需要一大批輔助伴侶蛋白的支持。為了找尋參與Hsp90-P復(fù)合物形成的關(guān)鍵輔助伴侶蛋白,我們首先分析了Hsp90變構(gòu)體抑制劑Celastrol對RABV病毒蛋白累積的影響。免疫印跡實(shí)驗(yàn)結(jié)果表明,Celastrol藥物具有類似17-AAG藥物的作用也能夠降低RABV P蛋白累積水平。由此提示輔助伴侶蛋白Cdc37可能參與了Hsp90-P復(fù)合物的形成。接著共聚焦實(shí)驗(yàn)顯示,RABV感染情況下含有P蛋白的NBs能夠大量募集Cdc37; Co-IP實(shí)驗(yàn)結(jié)果進(jìn)一步顯示Hsp90-Cdc37-P三元復(fù)合物的形成。由此表明,P蛋白是Hsp90/Cdc37伴侶系統(tǒng)的客戶蛋白。3.Cdc37募集P蛋白至Hsp90系統(tǒng)依賴和非依賴Hsp90結(jié)合途徑的發(fā)現(xiàn)已有研究報(bào)道Cdc37募集蛋白激酶至Hsp90系統(tǒng)存在依賴和非依賴Hsp90結(jié)合的途徑。而Cdc37對其他種類客戶蛋白的募集途徑不甚明了。那作為非蛋白激酶的RABVP蛋白,Cdc37對其募集至Hsp90系統(tǒng)是否也存在這兩條途徑或其他途徑?在本研究中我們證明Cdc37參與Hsp90-P復(fù)合物形成的四條途徑：缺失Hsp90結(jié)合區(qū)域的Cdc37截短體未能提升P蛋白的累積水平,而野生型Cdc37和Cdc37截短體Cdc37(aa 1-323)能夠通過依賴Hsp90結(jié)合的途徑募集P蛋白至Hsp90系統(tǒng)進(jìn)而促進(jìn)P蛋白的累積；破壞了與Hsp90結(jié)合能力的Cdc37M165和L206單點(diǎn)和雙點(diǎn)突變體依然保持著與P蛋白的互作,并提升P蛋白的累積水平,而且能夠模擬野生型Cdc37增強(qiáng)Hsp90-P之間的互作,由此指示野生型Cdc37還能夠以一種非依賴Hsp90結(jié)合的方式募集P蛋白至Hsp90系統(tǒng)來保護(hù)P蛋白的穩(wěn)定性：Cdc37的激活需要其Ser13位點(diǎn)的磷酸化修飾,非激活的Cdc37(將Cdc37 Ser13位點(diǎn)突變成Ala)并未減弱其與Hsp90和P蛋白的互作,而且能夠募集P蛋白至Hsp90系統(tǒng)進(jìn)而促進(jìn)P蛋白的累積,由此指示非激活的Cdc37存在類似于野生型Cdc37依賴Hsp90結(jié)合的方式募集P蛋白至Hsp90系統(tǒng)進(jìn)而對P蛋白進(jìn)行穩(wěn)定；破壞了與Hsp90結(jié)合能力的非激活Cdc37 M165和L206的單點(diǎn)和雙點(diǎn)突變體也依然保持著與P蛋白的互作,并提升P蛋白的累積水平,而且能夠模擬野生型Cdc37增強(qiáng)Hsp90-P之間的互作,由此指示非激活的Cdc37也能夠以一種非依賴Hsp90結(jié)合的方式募集P蛋白至Hsp90系統(tǒng)來保護(hù)P蛋白的穩(wěn)定性。4. Cdc37、Hsp90穩(wěn)定P蛋白正調(diào)控RABV感染的發(fā)現(xiàn)首先免疫印跡實(shí)驗(yàn)結(jié)果顯示RABV的感染能夠誘導(dǎo)Cdc37和Hsp90蛋白的表達(dá),由此提示Cdc37和Hsp90在RABV感染過程中可能起著正向調(diào)控作用。接著,采用真核過表達(dá)的方式研究Cdc37和Hsp90對RABV感染的影響,結(jié)果發(fā)現(xiàn),過表達(dá)Hsp90和Cdc37能夠從蛋白水平引起RABV病毒P蛋白的累積。然后我們通過免疫印跡實(shí)驗(yàn)發(fā)現(xiàn),過表達(dá)Cdc37和Hsp90能夠阻斷蛋白合成抑制劑CHX處理情況下的P蛋白降解,而對CHX處理情況下的N蛋白的降解無影響,由此表明Cdc37和Hsp90是從蛋白穩(wěn)定性水平而不是從蛋白合成水平影響RABV病毒P蛋白的累積。此外,采用RNAi干擾的方法降低Cdc37或Hsp90蛋白的表達(dá)能夠顯著降低病毒蛋白表達(dá)和病毒RNA合成的水平,并減少細(xì)胞內(nèi)病毒粒子的釋放量,由此證實(shí)了Cdc37和Hsp90在RABV感染的正向調(diào)控作用。最后,綜上數(shù)據(jù),Cdc37和Hsp90是通過穩(wěn)定P蛋白促進(jìn)P蛋白的累積進(jìn)而影響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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.65

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