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A High Capacity Assay for Inhibitors of Human Papillomavirus DNA Replication

Bio/Technology volume 13, pages 1210–1214 (1995)Cite this article

Abstract

The discovery of antiviral compounds against human papillomaviruses (HPV) has been hindered by the difficulties in culturing virus in vitro or assaying stable HPV DNA replication. However, plasmids containing the HPV replication origin replicate transiently upon co-transfection with HPV E1 and E2 expression vectors. We have adapted this assay using secreted alkaline phosphatase (SAP) as a reporter for rapid analysis of DNA copy number. Use of the S V40 early promoter in controlling SAP expression was critical in ensuring both a strong signal and copy number dependence: the stronger β-actin promoter inhibited replication, while the weaker SV40 late promoter yielded very low levels of SAP. The precise configuration of the E1 and E2 expression vectors also was critical, most pre-existing vectors did not support efficient replication and SAP secretion. The extent of DNA replication and SAP secretion were both proportional to the amount of E1/E2 vector used in transfections; under optimal conditions SAP increased 100-fold during replication. The assay has been developed for compound screening in 96-well plates and several inhibitors have been identified. Quantitative Southern blot analysis has shown that most of these inhibit HPV DNA replication rather than SAP accumulation or activity, and several are under test hi models of viral replication. The assay also provides a rapid system for functional analysis of the HPV E1, E2 genes and the replication origin.

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Author notes

  1. Michael A. Romanos: Corresponding author.

Authors and Affiliations

  1. Wellcome Research Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, UK

    Mary Plumpton, Nigel A. Sharp, Laura H. Liddicoat, Dale O. Tucker, Fiona J. Hughes, Stuart M. Russell & Michael A. Romanos

  2. Estonian Biocentre, Riia 23, EE2400, Tartu, Estonia

    Maido Remm

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  1. Mary Plumpton
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  2. Nigel A. Sharp
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Plumpton, M., Sharp, N., Liddicoat, L. et al. A High Capacity Assay for Inhibitors of Human Papillomavirus DNA Replication. Nat Biotechnol 13, 1210–1214 (1995). https://doi.org/10.1038/nbt1195-1210

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