title: Novel recombinant papillomavirus genomes expressing selectable genes.
abstract: Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 "marker" genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle.
authors.affiliation: Lab of Viral Diseases, NIAID, NIH, Bethesda, MD, USA.
authors.forename: Koenraad
authors.initials: K
authors.lastname: Van Doorslaer
authors.affiliation: Lab of Viral Diseases, NIAID, NIH, Bethesda, MD, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Porter
authors.affiliation: Lab of Viral Diseases, NIAID, NIH, Bethesda, MD, USA.
authors.forename: Caleb
authors.initials: C
authors.lastname: McKinney
authors.affiliation: Lab of Viral Diseases, NIAID, NIH, Bethesda, MD, USA.
authors.forename: Wesley H
authors.initials: WH
authors.lastname: Stepp
authors.affiliation: Lab of Viral Diseases, NIAID, NIH, Bethesda, MD, USA.
authors.forename: Alison A
authors.initials: AA
authors.lastname: McBride
mesh_terms: D003114:Colony-Forming Units Assay; D024882:Drug Resistance, Viral / Q000187:drug effects; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005814:Genes, Viral; D005819:Genetic Markers; D052161:Human papillomavirus 18 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D009355:Neomycin / Q000494:pharmacology; D011995:Recombination, Genetic / Q000235:genetics*; D016662:Virus Integration / Q000187:drug effects / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D005819:Genetic Markers; D009355:Neomycin