Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada.
Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA.
Virology. 2010 Mar 30;399(1):65-76. doi: 10.1016/j.virol.2009.12.026. Epub 2010 Jan 15.
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.
多瘤病毒和乳头瘤病毒的基因组复制分别由大 T 抗原 (LT) 和 E1 和 E2 的结合在病毒起源 (ori) 处启动。ori 含有质粒的复制发生在瞬时表达这些病毒蛋白的细胞中，通常通过 Southern 印迹或 PCR 进行定量。为了便于研究 SV40 和 HPV31 DNA 复制，我们开发了细胞测定法，其中使用位于 ori 顺式的萤火虫荧光素酶基因来定量 ori-质粒的瞬时复制。在优化条件下，SV40 和 HPV31 ori-质粒的复制分别导致萤火虫荧光素酶水平增加 50 倍和 150 倍。使用 LT、E1 和 E2 的复制缺陷突变体以及 DNA 复制和细胞周期进程抑制剂验证了这些结果。这些定量和高通量测定法应该极大地促进 SV40 和 HPV31 DNA 复制的研究以及该过程的小分子抑制剂的鉴定。
