Gauson Elaine J, Donaldson Mary M, Dornan Edward S, Wang Xu, Bristol Molly, Bodily Jason M, Morgan Iain M
VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, Virginia, USA University of Glasgow Institute of Infection, Immunology and Inflammation, Glasgow, United Kingdom.
University of Glasgow Institute of Infection, Immunology and Inflammation, Glasgow, United Kingdom.
J Virol. 2015 May;89(9):4980-91. doi: 10.1128/JVI.00335-15. Epub 2015 Feb 18.
To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination.
Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.
为了复制双链人乳头瘤病毒16型（HPV16）DNA基因组，病毒蛋白E1和E2与病毒复制起点结合，并且E2还可以调节相邻启动子的转录。E2与宿主蛋白相互作用以调节转录和复制；TopBP1和Brd4是与HPV16 E2相互作用的细胞蛋白。先前对E2突变体的研究表明，Brd4是E2反式激活特性所必需的，而TopBP1是E2与E1结合从病毒复制起点诱导DNA复制所必需的。最近的研究也表明Brd4参与了E2和E1对DNA复制的调节。在此，我们证明TopBP1和Brd4都存在于病毒复制起点，并且与E2的相互作用是DNA复制最佳起始所必需的。这两种细胞蛋白都存在于含有E1 - E2的核灶中，并且病毒复制起点是这些核灶有效形成所必需的。针对TopBP1或Brd4的短发夹RNA（shRNA）会破坏E1 - E2核体，但对E1 - E2介导的DNA复制水平没有影响。在完整HPV16基因组背景下的一个损害Brd4相互作用的E2突变体不能在原代人角质形成细胞中有效地建立游离型病毒基因组。总体而言，结果表明TopBP1与E2之间以及Brd4与E2之间的相互作用是正确启动DNA复制所必需的，但不是持续DNA复制所必需的，持续DNA复制可能由诸如滚环扩增和/或同源重组等替代过程介导。
人乳头瘤病毒16型（HPV16）是许多人类癌症的病原体，包括宫颈癌和头颈癌，并且在全球每年导致数十万人死亡。当前的疫苗将拯救后代的生命，但需要针对HPV16的抗病毒药物来减轻当代和后代的疾病负担。靶向由两种病毒蛋白E1和E2与诸如TopBP1和Brd4等细胞蛋白结合介导的病毒DNA复制将具有治疗益处。本报告表明这些细胞蛋白在HPV16 E1 - E2启动病毒DNA复制中起作用，但对持续复制不起作用。如果要有效靶向病毒复制，这一点很重要；我们需要了解病毒DNA复制每个阶段所需的病毒和细胞蛋白，以便能够有效地破坏它。
