cIAP2蛋白抑制乙型肝炎病毒复制的分子机制研究

发布时间：2018-01-13 00:34

  本文关键词：cIAP2蛋白抑制乙型肝炎病毒复制的分子机制研究　出处：《复旦大学》2011年博士论文　论文类型：学位论文

  更多相关文章： 乙型肝炎病毒 细胞凋亡抑制蛋白2 抗病毒活性 聚合酶 蛋白质降解 泛素化蛋白酶体系统 E3泛素连接酶 前基因组RNA包装

【摘要】：cIAP2蛋白抑制乙型肝炎病毒复制的分子机制研究 乙型肝炎病毒(Hepatitis B virus, HBV)是一种严重危害人类健康的病原体。已有的研究结果表明病毒和宿主的相互作用决定着病毒感染清除或持续性感染的建立。在HBV感染的清除过程中,机体可通过肿瘤坏死因子-α(Tumor necrosis factor alpha, TNF-α)、干扰素-γ(Interferon gamma, IFN-γ)、干扰素-α(Interferon alpha, IFN-α)、干扰素-β(Interferon beta, IFN-β)等细胞因子介导的非杀细胞方式抑制HBV复制,然而这些细胞因子调控的抗病毒基因尚未完全阐明。本室前期通过基因芯片等技术筛选TNF-α诱导的效应基因,发现细胞凋亡抑制蛋白2(cellular inhibitor of apoptosis protein 2, cIAP2)具有抑制HBV复制的效应,但其具体的抗病毒机制尚未完全阐明。 本研究首先在pCMV-HBV复制系统中验证cIAP2的抗病毒效应。研究结果显示,cIAP2并不影响HBV前基因组RNA的水平和衣壳蛋白的合成,但cIAP2显著下调了HBV复制中间体DNA的水平。siRNA干扰内源性cIAP2的表达后,转入HBV复制型质粒,检测内源性表达的cIAP2对HBV基因表达和复制的影响。研究结果显示,下调内源性cIAP2的表达可明显增强HBV的复制,而并不影响病毒转录的RNA和合成的衣壳蛋白。上述结果提示cIAP2可能影响了HBV前基因组RNA逆转录成DNA过程中的某个环节。 为明确cIAP2抑制HBV的功能区域,构建了cIAP2的不同缺失突变体。将HBV复制型质粒和cIAP2截短突变体共转细胞,检测病毒的基因表达和复制情况。研究结果显示,逐步缺失cIAP2蛋白N-端的三个BIR结构域,cIAP2的抗病毒效应有不同程度的削弱,但当缺失C-端的CARD和RING两个结构域后,cIAP2的抗病毒效应完全消失。已知RING结构域负责cIAP2的E3连接酶活性,进一步发现cIAP2的E3连接酶活性区域点突变(cIAP2*)后,其抑制HBV复制的作用完全消失。上述结果提示cIAP2对HBV复制的抑制作用依赖于其RING结构域的E3连接酶活性。 鉴于cIAP2抑制HBV复制与其E3连接酶活性相关,而E3连接酶可以促进蛋白的泛素化降解,推测cIAP2可能通过调控HBV复制关键蛋白的表达来发挥抑制HBV复制的效应。将HBV聚合酶、衣壳蛋白、HBx蛋白等表达质粒与cIAP2或cIAP2*表达质粒共转染细胞,筛查cIAP2是否能够调控HBV复制相关蛋白的表达。研究结果显示,cIAP2能够显著下调HBV聚合酶蛋白的表达水平,而不影响衣壳蛋白和HBx等蛋白的表达。cIAP2的E3连接酶活性突变体(cIAP2*)对聚合酶的表达无下调作用,提示cIAP2的E3连接酶活性对聚合酶的下调是必需的。为明确肝细胞内源性的cIAP2对聚合酶的表达是否具有下调作用,利用干扰RNA抑制内源性cIAP2的表达,再转入聚合酶蛋白表达质粒。结果显示,内源性cIAP2的表达被下调后,聚合酶蛋白的表达水平增强,证明内源性表达的cIAP2仍具有下调HBV聚合酶表达的作用。 蛋白的稳态表达水平由蛋白的合成和降解两方面决定。为排除cIAP2对聚合酶的下调是由于聚合酶高水平表达而发生错误折叠所致,本研究通过转染低剂量的质粒以降低聚合酶的表达水平,发现cIAP2对低水平表达的聚合酶仍具有明显的下调作用,对聚合酶蛋白相关伴侣分子Hsp90, Hsp70的表达并无影响,同时聚合酶蛋白的高水平表达未引起细胞未折叠蛋白反应。以上结果提示cIAP2并未影响聚合酶蛋白的合成,而可能是促进了聚合酶的降解。为此,进一步使用CHXchase方法检测聚合酶蛋白的半衰期。结果显示,cIAP2显著加快了聚合酶的降解速率,使聚合酶的半衰期由75分钟左右加快到25分钟左右,而cIAP2的E3连接酶活性缺失突变体(cIAP2*)不能缩短聚合酶蛋白的半衰期,相反可以延长聚合酶蛋白的半衰期。 细胞蛋白质的降解主要通过两种途径,溶酶体途径或蛋白酶体途径。为确定cIAP2降解聚合酶蛋白的可能途径,本研究分别引入两种途径的特异性抑制剂,检测其对cIAP2降解聚合酶的逆转作用。结果显示,cIAP2对聚合酶蛋白的降解可以被蛋白酶体抑制剂而非溶酶体抑制剂所逆转,提示cIAP2通过蛋白酶体途径降解聚合酶蛋白。鉴于cIAP2对聚合酶的降解与蛋白酶体活性相关且依赖其E3连接酶活性区域,推测cIAP2可能通过促进聚合酶的泛素化修饰来发挥降解作用。体内泛素化实验显示,cIAP2部分促进了聚合酶蛋白的泛素化修饰。以上结果提示cIAP2促进了聚合酶蛋白的泛素-蛋白酶体途径降解。 底物的泛素化修饰依赖底物和酶的相互作用,故推测聚合酶和cIAP2之间可能存在相互作用。体内免疫共沉淀和体外GST pull-down实验发现cIAP2和聚合酶之间存在相互作用,并且cIAP2蛋白N-端的BIR结构域,聚合酶的TP、RT、RH结构域参与了两者的结合。 聚合酶蛋白在HBV复制过程中结合前基因组RNA并与核心蛋白共同启动HBV前基因组RNA的包装,然后在核衣壳内合成HBV DNA。已经发现cIAP2参与降解聚合酶蛋白,推测cIAP2可能通过抑制HBV前基因组RNA包装来发挥抑制HBV复制的效应。研究结果显示,cIAP2下调了HBV核衣壳相关RNA的水平,且干扰cIAP2的表达后,HBV核衣壳相关RNA的水平显著增加,而HBV衣壳蛋白单体组装成核衣壳的过程未受影响,提示cIAP2下调聚合酶蛋白的表达后,直接影响了HBV前基因组RNA的包装。 总结上述结果,本研究发现cIAP2能够促进聚合酶的泛素-蛋白酶体途径降解,并通过下调HBV前基因组RNA的包装,发挥抑制HBV复制的作用。本研究进一步明确了TNF-α诱导的cIAP2的抗病毒作用及其分子机制,有助于解释TNF-α非杀细胞方式抑制HBV复制的机理。以聚合酶蛋白的泛素-蛋白酶体途径降解为切入点有望为新型抗乙肝药物的开发提供思路。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373.2

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