HPV16 E6 is necessary for the stable maintenance of HPV16 as an extrachromosomal genome.

<p>Shown are HPV16-specific Southern analyses of total genomic DNA isolated from early (A and B, passage 2) or late (C, passage 6 or higher) NIKS transfected with the indicated wild-type or E6 mutant HPV16 genomes (lanes labeled with the symbol “-” were transfected with the drug selection plasmid but not HPV16 DNA – see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003717#s2" target="_blank">methods</a>). For each sample, 20 µg of total genomic DNA was digested with XhoI (a noncutter of HPV16) prior to Southern analysis. Indicated at the left is the position of migration of open circular (OC), linear (LN) and supercoiled circular (SC) forms of HPV16 DNA. At the right side of panel B are copy number standards: 1.7 copies of HPV/cell from 20 µg DNA is equal to 43 pg of HPV. (A) HPV16 E6WT genomes replicated extrachromosomally as evidenced by the presence of open circular and supercoiled circular forms of the viral genome, whereas HPV16 E6STOP genomes were not detected. (B) HPV16 E6SAT was not maintained extrachromosomally as evidenced by the lack of open circular or supercoiled circular forms of HPV16 DNA. However, the HPV16 E6I128T and E6Δ146–151 mutant genomes replicated extrachromosomally with copy numbers comparable to the wild-type genome. In Southerns not shown, BamHI/DpnI digestions revealed that all of the detectable genomes were DpnI resistant. In (C) can be observed the continued maintenance of the HPV16 E6WT, E6I128T and E6Δ146–151 genomes as extrachromosomal nuclear plasmids in later passages of NIKS (passages 6 or higher). In (D) low molecular weight DNA (1.5*10⁶ cells worth) was digested with BamHI/DpnI. Unit length forms of HPV16 E6WT but not HPV16 E6*I and E6*II genomes could be detected. qPCR in (E) confirmed that the HPV16 E6WT genome was present at approximately 2.5 copies/cell while HPV16 E6*I and E6*II were present at .11 and 0.02 copies/cell respectively. Error bars represent the standard deviation.