Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication.
Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.
Fradet-Turcotte
Amélie
A
Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada.
Morin
Geneviève
G
Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada.
Lehoux
Michaël
M
Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada.
Bullock
Peter A
PA
Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA.
Archambault
Jacques
J
Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada. Electronic address: jacques.archambault@ircm.qc.ca.
D000957:Antigens, Viral, Tumor / Q000378:metabolism; D000998:Antiviral Agents / Q000494:pharmacology; D015139:Blotting, Southern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D005454:Fluorescence Polarization; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases; D027383:Papillomaviridae / Q000235:genetics / Q000502:physiology*; D011120:Polyomavirus / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D000093542:Gemcitabine
D000957:Antigens, Viral, Tumor; D000998:Antiviral Agents; D003841:Deoxycytidine; D008156:Luciferases; D000093542:Gemcitabine