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A novel drug screening assay for papillomavirus specific antiviral activity
P R Clark1, M L Roberts, L M Cowsert
1Department of Infectious Diseases, Isis Pharmaceuticals Inc., Carlsbad, CA 92008, USA.
Abstract:
Discovery and development of human papillomavirus (HPV) specific antiviral agents have been hampered by the lack of an in vitro assay permissive to HPV replication. An experimental assay system for monitoring HPV-11 DNA replication has been optimized for use as a papillomavirus antiviral drug screening tool. Cloned HPV DNA was introduced into SCC-4 cells by electroporation and viral DNA replication monitored by Southern blot. Kinetic studies demonstrated an increased HPV genome copy number with time. Viral DNA replicated as episomal, unit length genome and remained episomal after multiple passages. These data suggested the basis for an in vitro replication assay for evaluating the antiviral activity of potential chemotherapeutic agents directly on HPV. This model was used to investigate antiviral activities of current anti-HPV therapies such as 5-fluorouracil (5-FU) and alpha-interferon (alpha-IFN) and potential therapies such as sodium butyrate, 5-bromo-20-deoxyuridine (BrdU) and antisense oligonucleotides. HPV- 11 replication is significantly inhibited by BrdU and sodium butyrate; however 5-FU and alpha-IFN did not give consistent dose response results. Finally, ISIS 2105, a 20-mer phosphorothioate antisense oligonucleotide, which targets HPV-11 E2 gene product, showed potent antiviral activity in this assay with an IC50 of approximately 70 nM.
Insights
A new in vitro assay enables human papillomavirus (HPV) antiviral drug screening. This assay demonstrated that BrdU and sodium butyrate inhibit HPV-11 replication, while an antisense oligonucleotide showed potent antiviral activity.
Area of Science:
- Virology
- Antiviral Drug Discovery
Background:
- Human papillomavirus (HPV) antiviral agent development is hindered by the absence of a reliable in vitro replication assay.
- An optimized experimental system is crucial for screening potential HPV chemotherapeutics.
Purpose of the Study:
- To establish and validate an in vitro assay for monitoring HPV-11 DNA replication.
- To utilize this assay as a tool for screening antiviral agents against HPV.
Main Methods:
- Cloned HPV-11 DNA was introduced into SCC-4 cells via electroporation.
- Viral DNA replication was monitored using Southern blot analysis and kinetic studies.
- The assay was used to test the efficacy of 5-fluorouracil (5-FU), alpha-interferon (alpha-IFN), sodium butyrate, 5-bromo-20-deoxyuridine (BrdU), and an antisense oligonucleotide (ISIS 2105).
Main Results:
- The assay system successfully demonstrated HPV-11 DNA replication over time, with viral DNA remaining episomal.
- HPV-11 replication was significantly inhibited by BrdU and sodium butyrate.
- 5-FU and alpha-IFN did not yield consistent dose-response results.
- ISIS 2105, an antisense oligonucleotide targeting the HPV-11 E2 gene, exhibited potent antiviral activity with an IC50 of approximately 70 nM.
Conclusions:
- An in vitro replication assay for HPV-11 has been successfully developed and optimized.
- This assay serves as a valuable tool for evaluating the antiviral potential of various compounds.
- Antisense oligonucleotide ISIS 2105 demonstrates significant promise as an HPV-specific antiviral agent.