Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication

Abstract

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.

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

“…This method, developed by the Archambault laboratory for both BPV-1 and HPV-31 in C33a cells, requires the cotransfection of plasmids encoding E2, the homologous E1 helicase, Renilla luciferase (internal control), and firefly luciferase (target replicon) (41,42). The firefly luciferase gene is constitutively expressed due to the presence of a cytomegalovirus (CMV) promoter.…”

Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

“…Plasmid pFLORI11, which encodes the minimal HPV11 origin of DNA replication, was constructed by PCR amplification of the origin (nucleotides 7876 to 97 of the reference HPV11 genome obtained from ATCC; GenBank accession no. M14119) with a pair of primers containing synthetic NgoMIV restriction sites (underlined) (5=-CCAGCCGGCGTCACACACCTGCAAC-3= and 5=-CCCGCCGGCCCT CGTCTGCTAATTTTTTGG-3=), digestion of the amplified product with NgoMIV, and ligation of the digested DNA into the NgoMIV site of plasmid pCI-FLuc (22). The orientation of the origin fragment in pFLORI11 is such that nucleotide 7876 of the origin is closest to the simian virus 40 (SV40) polyadenylation site of pCI-FLuc.…”

“…The HPV11 DNA replication assay was developed and performed essentially as described previously for HPV31 and BPV1 (22,24,25). Briefly, 2.5 ϫ 10 4 C33A cells/well were seeded in white flat-bottom 96-well plates (Corning) and transfected, 24 h later, with the following four plasmids: for HPV11, p11E1 (10 ng), p11E2 (10 ng), pFLORI11 (2.5 ng), and the Renilla luciferase (RLuc) control plasmid pRL (0.5 ng) (22); for BPV1, pCG E1Eag 1235 Ϫ (10 ng; BPV1 E1 expression plasmid [23]), pBPV1E2 (10 ng [25]), pFLORI-BPV1 short (2.5 ng [25]), and pRL (0.5 ng). In all experiments, the total amount of transfected DNA was adjusted to 100 ng with pCI (Promega) as the carrier DNA.…”

“…2) (22,24,25). We chose the HPV11 system to evaluate the in vivo activity of the E1 CTD because it has proven to be more amenable to subsequent biochemical studies.…”

“…Upon introduction of these four plasmids into C33A cells, pFL11ORI is replicated to a higher copy number by E1 and E2, resulting in increased expression of the FLuc reporter gene. Hence, the ratio of the activities of FLuc and RLuc, which is typically measured 24 to 72 h posttransfection, provides a robust surrogate readout of the levels of HPV DNA replication that occurred during the time frame (22).…”