title: Interferon treatment of human keratinocytes harboring extrachromosomal, persistent HPV-16 plasmid genomes induces de novo viral integration.
abstract: Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss. However, treated cultures eventually gave rise to outgrowth of clones harboring integrated HPV-16 genomes expressing viral E6 and E7 oncogenes from chimeric virus-cell mRNAs similar to those in cervical and head and neck cancers. Surprisingly, every HPV-16 integrant that arose after IFN exposure stemmed from an independent integration event into a different cellular gene locus, even within parallel cultures started from small cell inocula and cultured separately for ≥ 25 doublings to permit the rise and expansion of spontaneous integrants. While IFN treatment conferred a growth advantage upon preexisting integrants added to mixed control cultures, our results indicate that IFN exposure directly or indirectly induces HPV-16 integration, rather than only selects preexisting, spontaneous integrants that appear to be much less frequent. We estimate that IFN exposure increased integration rates by ≥ 100-fold. IFN-induced HPV-16 integration involved a wide range of chromosomal loci with less apparent selection for recurrent insertions near genes involved in cancer-related pathways. We conclude that IFNs and other potential treatments targeting high-risk HPV persistence that disrupt viral genome replication may promote increased high-risk HPV integration as a step in cancer progression. Therapies against high-risk HPV persistence thus need to be evaluated for their integration-inducing potential.
authors.affiliation: Veterans Affairs Healthcare System and The Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA michael-lace@uiowa.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lace
authors.affiliation: Veterans Affairs Healthcare System and.
authors.forename: James R
authors.initials: JR
authors.lastname: Anson
authors.affiliation: Veterans Affairs Healthcare System and The Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Haugen
authors.affiliation: Veterans Affairs Healthcare System and.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Dierdorff
authors.affiliation: Veterans Affairs Healthcare System and The Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA.
authors.forename: Lubomir P
authors.initials: LP
authors.lastname: Turek
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D002472:Cell Transformation, Viral / Q000187:drug effects; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics; D005111:Extrachromosomal Inheritance; D016679:Genome, Viral / Q000187:drug effects*; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D015603:Keratinocytes / Q000187:drug effects* / Q000821:virology; D030361:Papillomavirus Infections / Q000235:genetics* / Q000821:virology; D010957:Plasmids / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D016662:Virus Integration / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D007371:Interferon-gamma