Tracking Replicating HPV Genomes in Proliferating Keratinocytes
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 ANCHORTM 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. ImportanceIn persistent HPV infection, the viral genome is maintained at a constant copy number, replicates in synchrony with host DNA during S-phase, and is partitioned into daughter cells. The exact method by which HPVs partition to daughter cells is not well understood and the elucidation of such mechanisms may reveal relevant pharmacological targets to combat persistent HPV infection.