Analysis of Human Papillomavirus Genome Replication Using Two‐ and Three‐Dimensional Agarose Gel Electrophoresis

Abstract

This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two-dimensional and neutral/neutral/alkaline three-dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard-to-interpret signals of human papilloma virus replication intermediates obtained from two- and three-dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple-dimension assays are assessed using previously published experimental data. © 2017 by John Wiley & Sons, Inc.

The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing

“…During the initial amplification phase of HPV DNA, it has been noticed that in addition to monomeric HPV genomes (containing a single copy of the HPV genome), the viral genomic oligomers also appear [26,27,36]. Furthermore, during the stable maintenance phase, the majority of HPV DNA molecules persist in the multimeric forms in U2OS cells [26,27].…”

“…U2OS cells (American Type Culture Collection, ATCC no: HTB-96), were grown in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum. U2OS cells were transfected by electroporation using a Bio-Rad Gene Pulser II apparatus equipped with a capacitance extender (Bio-Rad Laboratories) at 220V, and capacitance set to 975μF [36,37].…”

“…For the MfPV1 E1 mutant, the nucleotides (HindIII) were added after genomic position 887; for the MfPV1 E2 mutant, the nucleotides (BamHI) were added after genomic position 2549; for the MfPV5 E1 mutant, the nucleotides (HindIII) were added after genomic position 844; for the MfPV5 E2 mutant, the nucleotides (SacI) were added after genomic position 2702; for the MfPV8 E1 mutant, the nucleotides (SacI) were added after genomic position 2311; for the MfPV8 E2 mutant, the nucleotides (BglII) were added after genomic position 2784. The viral minicircle genomes were produced following the previously published protocol [36].…”

“…For transient replication assay of MfPV genomes, U2OS cells were transfected with 4–5 μg of the indicated MfPV minicircle genomes. Total genomic DNA [26] or low-molecular-weight (LMW) DNA was extracted at the indicated time points using the modified Hirt method [36,41]. Prior to analysis half of each extrachromosomal DNA sample extracted from a 100 mm cell culture dish or 5 μg of each total DNA sample was treated with specific endonucleases linearizing viral DNA and DpnI (Thermo Scientific) to remove bacterially produced input MfPV DNA.…”

“…Extracted DNA molecules were resolved in a 0.8% agarose gel in TAE (1× Tris-acetate-EDTA) buffer (0.8 V/cm for 20 h) and visualized via Southern blotting (previously described in ref. [36]). Hybridization probes were prepared from specific MfPV genomic fragments using the DecaLabel DNA Labeling Kit (Thermo Scientific) and radioactive [α-32P] dCTP (Hartmann Analytic).…”