Figure 4.
Efficient HPV replication requires an intact cyclin-binding motif and Cdk-phosphorylation sites in E1. (a and b) HPV replication assays were performed as described (18, 20). Low molecular weight DNA was analyzed by Southern blotting using labeled origin plasmid as a probe to detect the presence of DpnI-resistant replication products. This probe contains sequences that also hybridize with E1 and E2 plasmids. Blots were quantified by PhosphorImager analysis. E1* reflects the use of pMT2-E1* based plasmids, which express higher levels of protein and replication activity. (c) Levels of E1*, E1*ARA, and E1*ΔCdk proteins were determined by immunoblotting with anti-E1 antibodies. (d) Cell-free replication was conducted as described in Material and Methods and products detected by incorporation of [α-32P]dCTP.