DNA replicative functions of highly-expressed, codon-optimized human papillomavirus proteins E1 and E2.
Human papillomavirus (HPV) DNA replication requires functional HPV early (E) proteins E1 and E2. To determine the biological activity of HPV 16 E1 and E2 mutant proteins under consideration as vaccine candidates, we developed a sensitive real time PCR assay that monitors HPV origin-of-replication-driven DNA synthesis. The assay was used to determine the DNA replicative functions of highly-expressed, codon-optimized HPV 16 E1 and E2 wild type and mutant proteins in transient transfections. Under the assay conditions, the HPV 16 E1 mutant (W439R, G482D) did not support HPV origin-driven DNA synthesis. In contrast, however, an HPV 16 E2 mutant bearing an E39A substitution, reported previously to be severely compromised for DNA replication, was found to be reduced only two-fold in activity and, therefore, considered not sufficiently inactivated for use in vaccines that depend on endogenous protein expression.
Wang
Xin-Min
XM
Department of Microbial Vaccine Research, P O Box 4, West Point, PA 19486, USA.
Jansen
Kathrin U
KU
McClements
William L
WL
D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D003062:Codon / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology; D014773:Virology / Q000379:methods
D003062:Codon; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C093478:E1 protein, Human papillomavirus 16; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D014765:Viral Vaccines