本文關(guān)鍵詞：cIAP2蛋白抑制乙型肝炎病毒復(fù)制的分子機(jī)制研究　出處：《復(fù)旦大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乙型肝炎病毒 細(xì)胞凋亡抑制蛋白2 抗病毒活性 聚合酶 蛋白質(zhì)降解 泛素化蛋白酶體系統(tǒng) E3泛素連接酶 前基因組RNA包裝

【摘要】：cIAP2蛋白抑制乙型肝炎病毒復(fù)制的分子機(jī)制研究 乙型肝炎病毒(Hepatitis B virus, HBV)是一種嚴(yán)重危害人類健康的病原體。已有的研究結(jié)果表明病毒和宿主的相互作用決定著病毒感染清除或持續(xù)性感染的建立。在HBV感染的清除過程中,機(jī)體可通過腫瘤壞死因子-α(Tumor necrosis factor alpha, TNF-α)、干擾素-γ(Interferon gamma, IFN-γ)、干擾素-α(Interferon alpha, IFN-α)、干擾素-β(Interferon beta, IFN-β)等細(xì)胞因子介導(dǎo)的非殺細(xì)胞方式抑制HBV復(fù)制,然而這些細(xì)胞因子調(diào)控的抗病毒基因尚未完全闡明。本室前期通過基因芯片等技術(shù)篩選TNF-α誘導(dǎo)的效應(yīng)基因,發(fā)現(xiàn)細(xì)胞凋亡抑制蛋白2(cellular inhibitor of apoptosis protein 2, cIAP2)具有抑制HBV復(fù)制的效應(yīng),但其具體的抗病毒機(jī)制尚未完全闡明。 本研究首先在pCMV-HBV復(fù)制系統(tǒng)中驗(yàn)證cIAP2的抗病毒效應(yīng)。研究結(jié)果顯示,cIAP2并不影響HBV前基因組RNA的水平和衣殼蛋白的合成,但cIAP2顯著下調(diào)了HBV復(fù)制中間體DNA的水平。siRNA干擾內(nèi)源性cIAP2的表達(dá)后,轉(zhuǎn)入HBV復(fù)制型質(zhì)粒,檢測內(nèi)源性表達(dá)的cIAP2對HBV基因表達(dá)和復(fù)制的影響。研究結(jié)果顯示,下調(diào)內(nèi)源性cIAP2的表達(dá)可明顯增強(qiáng)HBV的復(fù)制,而并不影響病毒轉(zhuǎn)錄的RNA和合成的衣殼蛋白。上述結(jié)果提示cIAP2可能影響了HBV前基因組RNA逆轉(zhuǎn)錄成DNA過程中的某個環(huán)節(jié)。 為明確cIAP2抑制HBV的功能區(qū)域,構(gòu)建了cIAP2的不同缺失突變體。將HBV復(fù)制型質(zhì)粒和cIAP2截短突變體共轉(zhuǎn)細(xì)胞,檢測病毒的基因表達(dá)和復(fù)制情況。研究結(jié)果顯示,逐步缺失cIAP2蛋白N-端的三個BIR結(jié)構(gòu)域,cIAP2的抗病毒效應(yīng)有不同程度的削弱,但當(dāng)缺失C-端的CARD和RING兩個結(jié)構(gòu)域后,cIAP2的抗病毒效應(yīng)完全消失。已知RING結(jié)構(gòu)域負(fù)責(zé)cIAP2的E3連接酶活性,進(jìn)一步發(fā)現(xiàn)cIAP2的E3連接酶活性區(qū)域點(diǎn)突變(cIAP2*)后,其抑制HBV復(fù)制的作用完全消失。上述結(jié)果提示cIAP2對HBV復(fù)制的抑制作用依賴于其RING結(jié)構(gòu)域的E3連接酶活性。 鑒于cIAP2抑制HBV復(fù)制與其E3連接酶活性相關(guān),而E3連接酶可以促進(jìn)蛋白的泛素化降解,推測cIAP2可能通過調(diào)控HBV復(fù)制關(guān)鍵蛋白的表達(dá)來發(fā)揮抑制HBV復(fù)制的效應(yīng)。將HBV聚合酶、衣殼蛋白、HBx蛋白等表達(dá)質(zhì)粒與cIAP2或cIAP2*表達(dá)質(zhì)粒共轉(zhuǎn)染細(xì)胞,篩查cIAP2是否能夠調(diào)控HBV復(fù)制相關(guān)蛋白的表達(dá)。研究結(jié)果顯示,cIAP2能夠顯著下調(diào)HBV聚合酶蛋白的表達(dá)水平,而不影響衣殼蛋白和HBx等蛋白的表達(dá)。cIAP2的E3連接酶活性突變體(cIAP2*)對聚合酶的表達(dá)無下調(diào)作用,提示cIAP2的E3連接酶活性對聚合酶的下調(diào)是必需的。為明確肝細(xì)胞內(nèi)源性的cIAP2對聚合酶的表達(dá)是否具有下調(diào)作用,利用干擾RNA抑制內(nèi)源性cIAP2的表達(dá),再轉(zhuǎn)入聚合酶蛋白表達(dá)質(zhì)粒。結(jié)果顯示,內(nèi)源性cIAP2的表達(dá)被下調(diào)后,聚合酶蛋白的表達(dá)水平增強(qiáng),證明內(nèi)源性表達(dá)的cIAP2仍具有下調(diào)HBV聚合酶表達(dá)的作用。 蛋白的穩(wěn)態(tài)表達(dá)水平由蛋白的合成和降解兩方面決定。為排除cIAP2對聚合酶的下調(diào)是由于聚合酶高水平表達(dá)而發(fā)生錯誤折疊所致,本研究通過轉(zhuǎn)染低劑量的質(zhì)粒以降低聚合酶的表達(dá)水平,發(fā)現(xiàn)cIAP2對低水平表達(dá)的聚合酶仍具有明顯的下調(diào)作用,對聚合酶蛋白相關(guān)伴侶分子Hsp90, Hsp70的表達(dá)并無影響,同時聚合酶蛋白的高水平表達(dá)未引起細(xì)胞未折疊蛋白反應(yīng)。以上結(jié)果提示cIAP2并未影響聚合酶蛋白的合成,而可能是促進(jìn)了聚合酶的降解。為此,進(jìn)一步使用CHXchase方法檢測聚合酶蛋白的半衰期。結(jié)果顯示,cIAP2顯著加快了聚合酶的降解速率,使聚合酶的半衰期由75分鐘左右加快到25分鐘左右,而cIAP2的E3連接酶活性缺失突變體(cIAP2*)不能縮短聚合酶蛋白的半衰期,相反可以延長聚合酶蛋白的半衰期。 細(xì)胞蛋白質(zhì)的降解主要通過兩種途徑,溶酶體途徑或蛋白酶體途徑。為確定cIAP2降解聚合酶蛋白的可能途徑,本研究分別引入兩種途徑的特異性抑制劑,檢測其對cIAP2降解聚合酶的逆轉(zhuǎn)作用。結(jié)果顯示,cIAP2對聚合酶蛋白的降解可以被蛋白酶體抑制劑而非溶酶體抑制劑所逆轉(zhuǎn),提示cIAP2通過蛋白酶體途徑降解聚合酶蛋白。鑒于cIAP2對聚合酶的降解與蛋白酶體活性相關(guān)且依賴其E3連接酶活性區(qū)域,推測cIAP2可能通過促進(jìn)聚合酶的泛素化修飾來發(fā)揮降解作用。體內(nèi)泛素化實(shí)驗(yàn)顯示,cIAP2部分促進(jìn)了聚合酶蛋白的泛素化修飾。以上結(jié)果提示cIAP2促進(jìn)了聚合酶蛋白的泛素-蛋白酶體途徑降解。 底物的泛素化修飾依賴底物和酶的相互作用,故推測聚合酶和cIAP2之間可能存在相互作用。體內(nèi)免疫共沉淀和體外GST pull-down實(shí)驗(yàn)發(fā)現(xiàn)cIAP2和聚合酶之間存在相互作用,并且cIAP2蛋白N-端的BIR結(jié)構(gòu)域,聚合酶的TP、RT、RH結(jié)構(gòu)域參與了兩者的結(jié)合。 聚合酶蛋白在HBV復(fù)制過程中結(jié)合前基因組RNA并與核心蛋白共同啟動HBV前基因組RNA的包裝,然后在核衣殼內(nèi)合成HBV DNA。已經(jīng)發(fā)現(xiàn)cIAP2參與降解聚合酶蛋白,推測cIAP2可能通過抑制HBV前基因組RNA包裝來發(fā)揮抑制HBV復(fù)制的效應(yīng)。研究結(jié)果顯示,cIAP2下調(diào)了HBV核衣殼相關(guān)RNA的水平,且干擾cIAP2的表達(dá)后,HBV核衣殼相關(guān)RNA的水平顯著增加,而HBV衣殼蛋白單體組裝成核衣殼的過程未受影響,提示cIAP2下調(diào)聚合酶蛋白的表達(dá)后,直接影響了HBV前基因組RNA的包裝。 總結(jié)上述結(jié)果,本研究發(fā)現(xiàn)cIAP2能夠促進(jìn)聚合酶的泛素-蛋白酶體途徑降解,并通過下調(diào)HBV前基因組RNA的包裝,發(fā)揮抑制HBV復(fù)制的作用。本研究進(jìn)一步明確了TNF-α誘導(dǎo)的cIAP2的抗病毒作用及其分子機(jī)制,有助于解釋TNF-α非殺細(xì)胞方式抑制HBV復(fù)制的機(jī)理。以聚合酶蛋白的泛素-蛋白酶體途徑降解為切入點(diǎn)有望為新型抗乙肝藥物的開發(fā)提供思路。
[Abstract]:Study on the molecular mechanism of cIAP2 protein inhibition of hepatitis B virus replication
Hepatitis B virus (Hepatitis B, virus, HBV) is a serious hazard to human health pathogens. Studies have shown that the interaction between virus and host determines the clearance of the virus infection or persistent infection is established. In the elimination process of HBV infection in the body by tumor necrosis factor alpha (Tumor necrosis factor alpha TNF-, alpha interferon gamma (Interferon), gamma, IFN-, gamma interferon alpha (Interferon), alpha, IFN- alpha) and interferon beta (Interferon beta beta, IFN-) - kill cell cytokine mediated inhibition of HBV replication, but these antiviral genes regulating cytokines has not been fully elucidated. This previous by gene chip technology screening effect induced by TNF- gene, found that cell apoptosis inhibitory protein 2 (cellular inhibitor of apoptosis protein 2, cIAP2) inhibited HBV replication, but its specific resistance The mechanism of the virus has not been fully elucidated.
This study first verified the antiviral effect of cIAP2 in pCMV-HBV replication system. The results showed that cIAP2 did not affect the level of synthesis and capsid protein of genomic RNA HBV, but cIAP2 significantly lowered the expression level of endogenous cIAP2.SiRNA interference HBV replication intermediates DNA after replicating plasmid into HBV, detected the expression of endogenous cIAP2 the expression of HBV gene and replication. The results showed that the expression of endogenous cIAP2 can enhance HBV replication, but does not affect the viral transcription and synthesis of RNA capsid protein. These results suggest that cIAP2 may affect HBV genomic RNA by reverse transcription of a link in the process of DNA.
Inhibition of functional area of HBV specific cIAP2, constructed deletion mutant cIAP2. Copy the HBV plasmid and cIAP2 truncated mutants were cotransfected into cells, detect the expression of virus gene and replication. The results showed that the three BIR domain by deletion of cIAP2 protein N- end, antiviral effect of cIAP2 in different degree weakened, but when two domain deletion of C- terminal CARD and RING after the antiviral effect of cIAP2 disappeared. Known RING domain is responsible for cIAP2 E3 ligase activity, further found that the E3 ligase activity region cIAP2 mutation (cIAP2*), which inhibit HBV replication completely disappeared. These results suggest that cIAP2 of inhibition of HBV replication is dependent on its RING domain E3 ligase activity.
In view of the fact that cIAP2 inhibited HBV replication and E3 ligase activity, E3 ligase can promote the ubiquitination of proteins, that expression of cIAP2 may regulate HBV replication key proteins to exert the inhibitory effect on the replication of HBV. HBV polymerase, capsid protein, plasmid co transfection of plasmid and cIAP2 or cIAP2* expression HBx protein expression, screening whether cIAP2 can regulate HBV replication associated protein expression. The results showed that the expression level of cIAP2 could significantly downregulate HBV polymerase protein, but did not affect the expression of.CIAP2 capsid protein and HBx protein of the E3 is connected with the mutant enzyme activity (cIAP2*) had no effect on the expression of down polymerase, suggesting that cIAP2 E3 ligase activity is required the polymerase is reduced. Whether the expression of clear liver cells endogenous cIAP2 polymerase with downregulation of endogenous cIAP2 by RNA interference, the table Then, it was transferred into polymerase protein expression plasmid. The results showed that the expression level of polymerase protein was enhanced after the expression of endogenous cIAP2 was down regulated, indicating that the endogenous cIAP2 still has the function of downregulating HBV polymerase expression.
The expression level of protein homeostasis is decided by two aspects of protein synthesis and degradation. In order to exclude the cIAP2 of polymerase reduction is due to high levels of expression of polymerase errors caused by folding, the plasmid was transfected into low dose to reduce the expression level of polymerase, showed that the expression of cIAP2 on low level polymerase is obviously down regulated. The polymerase protein related molecular chaperone Hsp90, the expression of Hsp70 had no effect, while high levels of polymerase protein expression did not cause cell unfolded protein response. These results suggest that cIAP2 did not affect the synthesis of polymeric enzyme protein, which may promote the polymerase degradation. Therefore, the further use of CHXchase method for detection of polymerase protein half-life. The results showed cIAP2, significantly accelerate the degradation rate of the polymerase, the half-life of polymerase is accelerated by 75 minutes to 25 minutes The E3 ligase activity deletion mutant (cIAP2*) of cIAP2 could not shorten the half-life of PCR and, on the contrary, could prolong the half-life of PCR.
Degradation of cell proteins mainly through two ways, lysosomal pathway or proteasome pathway. In order to determine the possible degradation of cIAP2 polymerase protein, a specific inhibitor of this research were introduced in two ways, to detect the reversal effect on degradation of cIAP2 polymerase. The results showed that the degradation of the polymerase protein cIAP2 can be proteasome inhibitor the non lysosomal inhibitor reversed by cIAP2 via the proteasome degradation pathway of cIAP2 polymerase. Given the polymerase protein degradation and proteasome activity and its dependent E3 ligase activity region, suggesting that cIAP2 may play a role by promoting the degradation of ubiquitination. Polymerase showed that in vivo ubiquitination assay, cIAP2 promotes ubiquitination polymerase protein. These results suggest that cIAP2 promotes the degradation of protein by the ubiquitin proteasome pathway.
The substrate ubiquitination dependent interaction between substrate and enzyme, so that between the polymerase and cIAP2 may interact. Co immunoprecipitation and in vitro GST pull-down experiments showed that the interaction between cIAP2 and polymerase, and the BIR domain of cIAP2 protein by N- polymerase TP, RT and RH domains are involved in both combination.
Polymerase protein during HBV replication with genomic RNA and core protein and genomic RNA HBV jointly launched the package, and then in the synthesis of HBV DNA. has been found in the nucleocapsid protein involved in the degradation of cIAP2 polymerase, suggesting that cIAP2 may inhibit the HBV pregenome RNA package to play an inhibitory effect on the replication of HBV. The results showed that cIAP2 reduced HBV nucleocapsid related RNA level, and interfering with the expression of cIAP2, HBV related nucleocapsid RNA levels increased significantly, while the HBV capsid protein monomers assemble into nucleocapsid process was not affected, suggesting that the expression of cIAP2 protein after polymerase transfer, directly affect the packaging of genomic RNA before HBV.
Summing up the above results, this study found that cIAP2 can promote the degradation by the ubiquitin proteasome pathway, and through down-regulation of packaging of genomic RNA HBV, play a role in inhibition of HBV replication. This study further clarified the antiviral effect of TNF- induced by cIAP2 and its molecular mechanism helps explain TNF- alpha kill cell the inhibition mechanism of HBV replication. Provide ideas to the development of the polymerase protein degradation of the ubiquitin proteasome pathway as a starting point to new anti HBV drugs.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R373.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 邢同京 ,駱抗先 ,柴艷峰;干擾素γ和腫瘤壞死因子α對乙型肝炎病毒轉(zhuǎn)錄后調(diào)節(jié)序列功能的影響[J];中華醫(yī)學(xué)雜志;2002年21期

，

本文編號：1416719