Tracking replicating HPV genomes in proliferating keratinocytes

Authors

• ES Staedtler
• MJ Iadarola
• MR Sapio
• TS Williams
• E Frangos
• M Baƒçkonja
• AJ Mannes

Abstract

Human papillomavirus genomes replicate and partition as minichromosomes alongside host chromatin during persistent infection. We have previously shown that plasmids containing the HPV18 replication origin and viral transcriptional enhancer element can replicate stably in keratinocytes in the presence of the HPV18 genome. These small replicons express the neomycin resistance gene in both bacteria and eukaryotic cells and have minimal prokaryotic elements that could induce innate immunity. Additionally, the replicons are reliant on the presence of the viral genome for their own persistence. Here, we use the replicons as vectors to express different fluorescent proteins that are detectable solely in the company of the viral genome. To generate an optimal expression cassette, we have identified enhancers, promoters and polyadenylation sites that function well in keratinocytes, and have selected fluorescent proteins that are detectable in proliferating living cells. This molecular tool allows us to indirectly monitor the presence of the virus in live cells and can inform on models of papillomavirus extrachromosomal genome maintenance, tethering, and amplification.

Scientific Focus Area: Virology

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