RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.
AscentGene, Inc., Gaithersburg, Maryland, USA.
mBio. 2017 May 30;8(3):e00713-17. doi: 10.1128/mBio.00713-17.
The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter.
人乳头瘤病毒（HPV）的生命周期与角质形成细胞分化密切相关。尽管病毒早期基因的表达在病毒感染未分化的基底细胞后立即启动，但病毒 DNA 的扩增和晚期基因的表达仅发生在经历终末分化的角质形成细胞的中到上层。在本报告中，我们表明 HPV18 无 TATA 晚期启动子 P 的相对活性取决于其相对于病毒 DNA 复制原点（Ori）的方向，并且对真核 DNA 聚合酶抑制剂 aphidicolin 敏感。此外，转染与 Ori 附近区域同源的 70 个核苷酸（nt）长的单链 DNA 寡核苷酸可诱导晚期启动子活性。我们还发现，筏培养物中的启动子激活导致产生晚期启动子相关的、有意义链转录起始 RNA（tiRNA）和剪接位点小 RNA（spliRNA）。最后，鉴定了一个作为启动子抑制剂的 -ACTING AAGTATGCA 核心元件。该元件与 hnRNP D0B 和 hnRNP A/B 因子相互作用。核心内的点突变阻止了 hnRNP 的结合并增加了启动子活性。证实了这一结果，在角质形成细胞中敲低两种 hnRNP 的表达导致启动子活性增加。综合这些数据，我们的研究揭示了 HPV18 晚期启动子如何受 DNA 复制和宿主因子调控的机制。几十年来，几乎所有 DNA 病毒的晚期启动子活性都与病毒 DNA 复制相关。然而，DNA 复制如何激活病毒晚期启动子以及复制机制的哪些成分参与其中，在很大程度上仍然未知。在这项研究中，我们对 HPV18 的 P 启动子区域进行了表征，并证明其激活依赖于 DNA 复制的方向。使用针对前导链或滞后链复制叉的单链寡核苷酸，我们表明病毒滞后链复制激活了启动子。我们还鉴定了位于启动子转录起始位点上游的转录抑制元件，该元件与细胞蛋白 hnRNP D0B 和 hnRNP A/B 相互作用，并调节晚期启动子活性。这是关于 DNA 复制如何激活病毒晚期启动子的第一个报告。
