Control mechanisms of productive papillomavirus replication by the viral E8^E2 protein

高危型人乳头瘤病毒（HPV）感染可导致上皮内病变，进而发展为浸润性癌症。HPV将其复制周期与感染的角质形成细胞的分化状态联系起来。HPV在上皮基底层的角质形成细胞中建立非生产性的持续感染。在基底层上，受感染的细胞开始其分化程序，但也重新进入细胞周期并激活DNA复制。然后，启动生产性复制，其特征在于病毒基因组的扩增和驱动病毒E4蛋白和病毒衣壳蛋白表达的病毒晚期启动子的激活。尽管有深入的研究，但分化细胞中基因组扩增和晚期启动子诱导的机制还不是很清楚，保守的HPV E8 ^E2转录因子是未分化细胞中病毒复制和基因表达的有效阻遏物，但其对PV的进化优势尚不清楚。在上一个资助期使用小家鼠PV 1（MmuPV 1）-小鼠模型获得的结果显示，MmuPV 1 E8 ^E2蛋白也是病毒基因表达的有效阻遏物，与HPV相同，使用细胞NCoR/SMRT辅阻遏物复合物进行阻遏。令人惊讶的是，E8^E2敲除（ko）或NCoR/SMRT结合缺陷基因组不能诱导T细胞缺陷型Foxn 1 nu/nu小鼠的尾疣或生殖道感染。E8 → E2 mt基因组极大地提高了表达晚期病毒E4蛋白的未分化角质形成细胞的分数，这导致G2细胞周期停滞。这很可能干扰感染小鼠基底层中MmuPV 1 E8 ~ E2 mt感染的角质形成细胞的分裂和扩增，从而干扰疣的形成。与E8^E2主要抑制生产性复制的观点一致，我们还发现在器官型培养物中由HPV 16 E8^E2 ko mt基因组诱导的细胞转录组的变化与病毒晚期基因表达的程度相关。针对HPV 16 E2蛋白的新型抗体揭示，E8^E2抑制核病毒E2灶的形成，核病毒E2灶最有可能代表病毒复制中心，并且这些细胞表达E4蛋白。这有力地表明，E8^E2的主要功能是防止E2蛋白在未分化细胞中积累，这将导致病毒基因组扩增、晚期启动子诱导和E4表达。基于这些结果，我们计划确定生产PV复制诱导的宿主转录组变化，并解决生产复制的选定基因的功能相关性。此外，我们计划探索E8^E2控制E2积累从而控制生产性复制的机制。总之，该项目旨在确定控制生产PV复制的机制，这可能有助于找到抗PV治疗的保守靶点。

Infections with high risk human papillomaviruses (HPV) can result in intraepithelial lesions that can progress to invasive cancers. HPV have linked their replication cycle to the differentiation state of the infected keratinocyte. HPV establish a non-productive, persistent infection in keratinocytes in the basal layer of the epithelium. In suprabasal layers, infected cells start their differentiation program, but also re-enter the cell cycle and activate DNA replication. Then, productive replication is initiated which is characterized by the amplification of viral genomes and activation of the viral late promoter which drives expression of the viral E4 protein and the viral capsid proteins. Despite intensive research, the mechanisms responsible for genome amplification and late promoter induction in differentiated cells are not very well understood.The conserved HPV E8^E2 transcription factor is a potent repressor of viral replication and gene expression in undifferentiated cells, but its evolutionary advantage for PV is not understood. Results obtained in the previous funding period using the Mus musculus PV1 (MmuPV1)-mouse model have revealed that the MmuPV1 E8^E2 protein is also a potent repressor of viral gene expression and, identical to HPV, uses cellular NCoR/SMRT corepressor complexes for repression. Surprisingly, E8^E2 knock-out (ko) or NCoR/SMRT-binding deficient genomes are unable to induce tail warts or manifest infections of the genital tract of T-cell deficient Foxn1nu/nu mice. E8^E2 mt genomes greatly enhance the fraction of undifferentiated keratinocytes expressing the late viral E4 protein and this results in a G2 cell cycle arrest. This most likely interferes with the division and, thus, expansion of MmuPV1 E8^E2 mt infected keratinocytes in the basal layer in infected mice and therefore wart formation. Consistent with the idea that E8^E2 mainly represses productive replication, we also find that changes of the cellular transcriptome induced by a HPV16 E8^E2 ko mt genome in organotypic cultures correlate with the extent of viral late gene expression. Novel antibodies against the HPV16 E2 protein reveal that E8^E2 inhibits the formation of nuclear viral E2 foci which, most likely, represent viral replication centers and that such cells express E4 protein. This strongly suggests that the main function of E8^E2 is to prevent E2 protein accumulation in undifferentiated cells which would result in viral genome amplification, late promoter induction and E4 expression. Based on these results, we plan to identify host transcriptome changes induced by productive PV replication and address the functional relevance of selected genes for productive replication. Furthermore, we plan to explore the mechanisms by which E8^E2 controls E2 accumulation and thus productive replication. In summary, this project aims to identify mechanisms controlling productive PV replication which may help to find conserved targets for anti-PV therapy.