Shin Jonathan R, Avilov Ivan, Schelhaas Mario, Gallardo Franck, McBride Alison A
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Institute of Cellular Virology, University of Münster, Münster, Germany.
bioRxiv. 2025 Mar 19:2025.03.18.644043. doi: 10.1101/2025.03.18.644043.
Human papillomavirus (HPV) genomes replicate and partition as minichromosomes alongside host chromatin during persistent infection. However, it is difficult to monitor genome dynamics in living cells because the small and compact genome will not easily tolerate expression cassettes. Here, we use ANCHOR technology to detect HPV18 genomes in living cells. We incorporated the cis element from ANCHOR technology into the late region of the HPV18 genome and expressed the ParB-GFP protein from an HPV18-dependent replicon. The replicon contains the HPV18 replication origin and viral transcriptional enhancer element and can replicate stably in keratinocytes when complemented by the HPV18 genome. This small replicon expresses the neomycin resistance gene in both bacteria and eukaryotic cells and has minimal prokaryotic elements that could induce innate immunity. This molecular tool enables us to indirectly monitor the presence of the virus by detecting these fluorescent proteins in live cells and allows for real-time tracking of replicating HPV18-ANCH3 genomes in proliferating keratinocytes to inform on models of HPV genome maintenance, tethering, and amplification. Here, we visualize partitioning of the viral DNA in dividing cells and show that HPV18-ANCH genomes are distributed somewhat equally to daughter cells by random attachment to host chromosomes.
在持续性感染期间，人乳头瘤病毒（HPV）基因组作为微型染色体与宿主染色质一起复制和分离。然而，由于小型紧凑的基因组不易容纳表达盒，因此难以监测活细胞中的基因组动态。在这里，我们使用锚定（ANCHOR）技术来检测活细胞中的HPV18基因组。我们将来自锚定技术的顺式元件整合到HPV18基因组的晚期区域，并从依赖HPV18的复制子表达ParB-GFP蛋白。该复制子包含HPV18复制起点和病毒转录增强元件，当由HPV18基因组补充时可在角质形成细胞中稳定复制。这个小复制子在细菌和真核细胞中均表达新霉素抗性基因，并且具有可诱导先天免疫的最小原核元件。这种分子工具使我们能够通过检测活细胞中的这些荧光蛋白间接监测病毒的存在，并允许实时跟踪增殖角质形成细胞中复制的HPV18-ANCH3基因组，为HPV基因组维持、拴系和扩增模型提供信息。在这里，我们可视化了分裂细胞中病毒DNA的分离，并表明HPV18-ANCH基因组通过随机附着于宿主染色体而在子细胞中大致平均分配。
