A HIGH-CAPACITY ASSAY FOR INHIBITORS OF HUMAN PAPILLOMAVIRUS DNA-REPLICATION

The discovery of antiviral compounds against human papillomaviruses (H PV) has been hindered by the difficulties in culturing virus in vitro or assaying stable HPV DNA replication, However, plasmids containing t he HPV replication origin replicate transiently upon co-transfection w ith 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 SV40 early promoter in controlling SAP expression was critical in ensuring both a strong signal and copy numb er dependence: the stronger beta-actin promoter inhibited replication, while the weaker SV40 late promoter yielded very low levels of SAP, T he precise configuration of the E1 and E2 expression vectors also was critical, most pre-existing vectors did not support efficient replicat ion and SAP secretion. The extent of DNA replication and SAP secretion were both proportional to the amount of E1/E2 vector used in transfec tions; under optimal conditions SAP increased 100-fold during replicat ion. The assay has been developed for compound screening in 96-well pl ates and several inhibitors have been identified. Quantitative Souther n blot analysis has shown that most of these inhibit HPV DNA replicati on rather than SAP accumulation or activity, and several are under tes t in models of viral replication, The assay also provides a rapid syst em for functional analysis of the HPV E1, E2 genes and the replication origin.