The mechanism of the inhibition of the replication of carcinogenic human papillomaviruses by the NCOR/SMRT-repressor complex

高风险（HR）人乳头瘤病毒（HPV）16、18、31等的持续感染导致人类的肛门生殖器癌和口咽癌。此外，来自β属的HPV感染与非黑色素瘤皮肤癌的发展有关。病毒E2蛋白家族控制病毒癌基因的转录和基因组的复制。序列分析表明，除了E2复制激活蛋白外，所有HPV都可以表达E8-E2 C蛋白。HPV 16和31 E8-E2 C蛋白已被证明是持续期病毒复制的关键负调节因子。第一个资助期的结果首次表明，E8^E2C的功能在HPV中高度保守，因为HPV 1（一种mu-PV）和HPV 8（一种beta-PV）的E8^E2C敲除基因组在人类角质形成细胞中过度复制。来自第一个资助期的已发表结果表明，E8^E2C令人惊讶地也限制了HPV 16的生产性复制。此外，我们还发现HPV 16 E8^E2C由E1基因内的一个新启动子转录，该启动子受宿主细胞和病毒蛋白的正调控和负调控。E8 ~ E2 C蛋白具有两个保守结构域：E8结构域负责抑制病毒转录和基因组复制，而E2 C部分介导特异性DNA结合和二聚化。第一个资助期的结果表明，转录和DNA复制的抑制是HPV E8阻遏物结构域与细胞NCoR/SMRT共阻遏物复合物之间保守相互作用的结果，该复合物由GPS 2、HDAC 3、NCoR、SMRT、TBL 1和TBLR 1蛋白组成。这首次表明NCoR/SMRT复合物不仅控制细胞基因表达，而且还抑制HPV复制。第一个资助期的结果表明，E8^E2C将NCoR/SMRT复合物募集到病毒复制起点，干扰了细胞复制蛋白与病毒E1解旋酶的结合，这很可能有助于抑制复制。本项目的目的是详细研究病毒复制激活蛋白与细胞复制蛋白在体外和体内E8^E2C存在和不存在的情况下的相互作用，以阐明NCoR/SMRT介导的病毒复制抑制的分子机制。此外，还将研究E8、E2 C-NCoR/SMRT相互作用对宿主细胞基因表达和HPV生命周期中DNA损伤途径的影响。该研究的长期目标是确定抗HPV治疗的新靶点。

Persistent infections with high-risk (HR) human papillomaviruses (HPV) 16, 18, 31 etc. cause ano-genital and oro-pharyngeal cancers in humans. Furthermore, infections with HPV from the genus beta have been implicated in the development of non-melanoma skin cancer. The viral E2 protein family controls transcription of the viral oncogenes and the replication of the genomes. Sequence analyses indicate that all HPV can express an E8^E2C protein in addition to the E2 replication activator protein. The HPV16 and 31 E8^E2C proteins have been shown to be crucial, negative regulators of the viral replication in the persistent phase. Results of the first funding period show for the first time that E8^E2C´s function is highly conserved among HPV as E8^E2C knock-out genomes of HPV1, a mu-PV and HPV8, a beta-PV over replicate in human keratinocytes. Published results from the first funding period demonstrate that E8^E2C surprisingly also limits the productive replication of HPV16. In addition, we also found that HPV16 E8^E2C is transcribed from a novel promoter within the E1 gene that is positively and negatively regulated by host cell and viral proteins. E8^E2C proteins have two conserved domains: the E8 domain is responsible for the inhibition of viral transcription and genome replication whereas the E2C part mediates specific DNA-binding and dimerization. Results from the first funding period indicate that the inhibition of transcription and DNA replication is the consequence of a conserved interaction between the HPV E8 repressor domain and the cellular NCoR/SMRT co-repressor complex, which consists of the GPS2, HDAC3, NCoR, SMRT, TBL1 and TBLR1 proteins. This suggests for the first time that the NCoR/SMRT complex not only controls cellular gene expression but also inhibits HPV replication. Results from the first funding period show that the recruitment of the NCoR/SMRT complex by E8^E2C to the viral replication origin interferes with the binding of cellular replication proteins to the viral E1 helicase which most likely contributes to the inhibition of replication. Aims of the project are to investigate in detail the interactions of viral replication activator proteins with cellular replication proteins in vitro and in vivo in the absence and presence of E8^E2C in order to elucidate the molecular mechanism of the NCoR/SMRT-mediated inhibition of viral replication. Furthermore, the influence of the E8^E2C-NCoR/SMRT interaction on host cell gene expression and the DNA damage pathway during the HPV life cycle will be investigated. The long-term goal of the study is the identification of novel targets for anti-HPV therapy.