UNIT 14B.1 Cell Culture Assay for Transient Replication of Human and Animal Papillomaviruses

This unit contains protocols for evaluation of replication functionality of papillomavirus genomes or subgenomic fragments. Replication is measured after transient cotransfection of the genome (or subgenomic fragment) with expression vectors encoding the viral E1 and E2 proteins. Input DNA is methylated at the adenine of GATC sequences by propagation in E. coli. DNA that replicates in mammalian cells will lose the adenine methylation and become DpnI-resistant, while residual methylated input DNA will remain DpnI-sensitive. After transfection, DNA extraction, and DpnI digestion, replicated DNA can be detected by Southern blotting as a full-length plasmid, since it is resistant to digestion. This assay can be used to map the genomic location of a functional origin or to evaluate replication activity of mutations in either the origin DNA sequences or the E1 or E2 proteins. Curr. Protoc. Microbiol. 24:14B.1.1-14B.1.18. © 2012 by John Wiley & Sons, Inc. Keywords: replication; transfection; HIRT extraction; Southern blot; papillomaviruses