人巨细胞病毒蛋白pUL23影响宿主蛋白RACK1与STAT1间的相互作用
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摘要
人巨细胞病毒（Human cytomegalovirus HCMV）属疱疹病毒科β亚属（β疱疹病毒）,在人群中的感染率十分普遍。HCMV像其它β疱疹病毒一样，不能被宿主体内的免疫系统完全清除，它会以一种较低的病毒水平保持对宿主的持续感染或者是以一种非活性状态持续潜伏于宿主体内，因而对于免疫功能正常的人，HCMV可长期潜伏而不致病。但是当宿主免疫功能低下时，HCMV则可大量繁殖，进而引起严重的致死并发症。随着目前多个HCMV病毒株完整基因组核酸序列的分析完成，HCMV基因组结构已基本被阐明。通过对病毒临床株基因的分析和缺失突变等发现，在200多个开放阅读框中（Open Reading Frame，ORF），仅有45到57个ORFs为病毒在人成纤维细胞中繁殖所必要，余下的都为非必需基因。研究表明，部分病毒蛋白参与了协调与宿主间细胞关系，以达到自身与宿主细胞长期共生的目的。
UL23基因是HCMV US22基因家族的成员，它能编码一个大小为33kD的病毒皮层蛋白pUL23,聚集在细胞质中的核周边区域。然而对于pUL23蛋白的功能目前了解甚少。为了进一步阐明pUL23的潜在功能，本实验室前期工作利用酵母双杂交方法，从人胚肾cDNA文库中筛选到多个与pUL23相互作用的宿主蛋白。RACK1[Receptor for activated C kinase1]就是其中之一。RACK1是蛋白激酶C的受体蛋白，因其可以和多种类型的蛋白分子相结合，从而被普遍地认为是一种多功能脚手架蛋白。本课题利用回复性酵母双杂交、免疫共沉淀等实验方法进一步确认了pUL23与RACK1间的相互作用,且pUL23和RACK1共定位于细胞质中。
研究发现RACK1能够与非磷酸化的STAT1结合，并作为一个脚手架蛋白发挥了招募STAT1到干扰素受体上的功能。而STAT1与干扰素受体的预先结合是STAT1活化和干扰素信号途径传导必不可少的一步，这说明RACK1与STAT1的结合对STAT1的活化和干扰素信号途径的传导是至关重要的。因此本文利用GSTpull-down实验检测了pUL23与RACK1的结合对RACK1-STAT1相互结合的影响，实验结果表明pUL23减弱了RACK1与STAT1间的亲和力，这说明病毒蛋白pUL23能够更牢固的“抓住”RACK1蛋白，进而导致RACK1、STAT1或许还有干扰素受体在内的复合物的解离。由文献可知，RACK1与STAT1结合的减弱会直接导致STAT1磷酸化水平的下降和干扰素抗病毒信号途径传导的受阻。因此本文的研究结果提高了pUL23在干扰素信号途径中发挥调节作用的可能性，并为今后pUL23蛋白功能的研究提供了诸多有意义的线索。
Human cytomegalovirus (HCMV) is the prototypical member of thebetaherpesvirus family and is a ubiquitous human pathogen. HCMV, like otherherpesviruses, cannot be completely eliminated by the immune system and remainseither as a low-level persistent infection or in a quiescent latent state for the lifetimeof the infected person. Therefore HCMV infections in immunocompetent adults areusually benign and could maintain a lifelong relationship with its host, butreactivation of HCMV from latency causes life-threatening illness in immunologicallyimmature or compromised individuals. According to the global analysis of severalHCMV strains genome, it revealed HCMV contains more than200ORFS (OpenReading Frame, ORF), but only45~57ORFS of them are essential for HCMV growthin primary human fibroblasts, the rest are nonessential. Studies have revealed someHCMV proteins have involved in coordinating the relationship with the host cell bytheir interaction with host cell protein.
UL23is a member of HCMV US22gene family, it encodes a tegument proteinpUL23about33kD which is located in cytoplasmic protein aggregates. But thebiological function of pUL23is controversial and has been not clear to this day. Toinvestigate the underlying function of pUL23, the yeast two-hybrid screening systemand co-immunoprecipitation (Co-IP) experiment were used to identify cellular targetsof pUL23. One of the binding partners of pUL23was RACK1, which is the foundingmember of the family of receptors for activated C kinase (PKC) collectively calledRACKs and is identified as a multifunctional scaffold protein. Furthermore pUL23and RACK1substantially co-localized in the cytoplasm when they wereco-transformed into Hela cells.
RACK1and has been reported to function as an adaptor recruiting thetranscription factor STAT1to the receptor complex in the IFN system, and thispre-association of STAT1with the receptor is required for STAT1activation and IFNsignaling. Therefore it's suggested the interaction between RACK1and STAT1plays a central role in STAT1activation and IFN signaling. Based on these clues, we exploredwhether HCMV pUL23may influence the interaction between RACK1and STAT1with RACK1as a mediator. Glutathione S-transferase pull-down experiment revealedthat the association of RACK1with HCMV protein pUL23with a higher affinity thanSTAT1. Therefore, it is suggested that the viral protein pUL23has the ability tointeract strongly with RACK1and consequently to bring about the disruption of thecomplex formed from STAT1, RACK1, and probably the IFN receptor, which maydirectly lead to the suppression of STAT1activation. While the activation of STAT1isimportant for IFN signaling pathway. Therefore, these findings raise the possibilitythat pUL23functions as a regulator in IFN signaling and provide us with manyimportant clues for the further research of pUL23.
UL23基因是HCMV US22基因家族的成员，它能编码一个大小为33kD的病毒皮层蛋白pUL23,聚集在细胞质中的核周边区域。然而对于pUL23蛋白的功能目前了解甚少。为了进一步阐明pUL23的潜在功能，本实验室前期工作利用酵母双杂交方法，从人胚肾cDNA文库中筛选到多个与pUL23相互作用的宿主蛋白。RACK1[Receptor for activated C kinase1]就是其中之一。RACK1是蛋白激酶C的受体蛋白，因其可以和多种类型的蛋白分子相结合，从而被普遍地认为是一种多功能脚手架蛋白。本课题利用回复性酵母双杂交、免疫共沉淀等实验方法进一步确认了pUL23与RACK1间的相互作用,且pUL23和RACK1共定位于细胞质中。
研究发现RACK1能够与非磷酸化的STAT1结合，并作为一个脚手架蛋白发挥了招募STAT1到干扰素受体上的功能。而STAT1与干扰素受体的预先结合是STAT1活化和干扰素信号途径传导必不可少的一步，这说明RACK1与STAT1的结合对STAT1的活化和干扰素信号途径的传导是至关重要的。因此本文利用GSTpull-down实验检测了pUL23与RACK1的结合对RACK1-STAT1相互结合的影响，实验结果表明pUL23减弱了RACK1与STAT1间的亲和力，这说明病毒蛋白pUL23能够更牢固的“抓住”RACK1蛋白，进而导致RACK1、STAT1或许还有干扰素受体在内的复合物的解离。由文献可知，RACK1与STAT1结合的减弱会直接导致STAT1磷酸化水平的下降和干扰素抗病毒信号途径传导的受阻。因此本文的研究结果提高了pUL23在干扰素信号途径中发挥调节作用的可能性，并为今后pUL23蛋白功能的研究提供了诸多有意义的线索。
Human cytomegalovirus (HCMV) is the prototypical member of thebetaherpesvirus family and is a ubiquitous human pathogen. HCMV, like otherherpesviruses, cannot be completely eliminated by the immune system and remainseither as a low-level persistent infection or in a quiescent latent state for the lifetimeof the infected person. Therefore HCMV infections in immunocompetent adults areusually benign and could maintain a lifelong relationship with its host, butreactivation of HCMV from latency causes life-threatening illness in immunologicallyimmature or compromised individuals. According to the global analysis of severalHCMV strains genome, it revealed HCMV contains more than200ORFS (OpenReading Frame, ORF), but only45~57ORFS of them are essential for HCMV growthin primary human fibroblasts, the rest are nonessential. Studies have revealed someHCMV proteins have involved in coordinating the relationship with the host cell bytheir interaction with host cell protein.
UL23is a member of HCMV US22gene family, it encodes a tegument proteinpUL23about33kD which is located in cytoplasmic protein aggregates. But thebiological function of pUL23is controversial and has been not clear to this day. Toinvestigate the underlying function of pUL23, the yeast two-hybrid screening systemand co-immunoprecipitation (Co-IP) experiment were used to identify cellular targetsof pUL23. One of the binding partners of pUL23was RACK1, which is the foundingmember of the family of receptors for activated C kinase (PKC) collectively calledRACKs and is identified as a multifunctional scaffold protein. Furthermore pUL23and RACK1substantially co-localized in the cytoplasm when they wereco-transformed into Hela cells.
RACK1and has been reported to function as an adaptor recruiting thetranscription factor STAT1to the receptor complex in the IFN system, and thispre-association of STAT1with the receptor is required for STAT1activation and IFNsignaling. Therefore it's suggested the interaction between RACK1and STAT1plays a central role in STAT1activation and IFN signaling. Based on these clues, we exploredwhether HCMV pUL23may influence the interaction between RACK1and STAT1with RACK1as a mediator. Glutathione S-transferase pull-down experiment revealedthat the association of RACK1with HCMV protein pUL23with a higher affinity thanSTAT1. Therefore, it is suggested that the viral protein pUL23has the ability tointeract strongly with RACK1and consequently to bring about the disruption of thecomplex formed from STAT1, RACK1, and probably the IFN receptor, which maydirectly lead to the suppression of STAT1activation. While the activation of STAT1isimportant for IFN signaling pathway. Therefore, these findings raise the possibilitythat pUL23functions as a regulator in IFN signaling and provide us with manyimportant clues for the further research of pUL23.
引文
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