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Updated: Apr 25, 2026
Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment
Published on: March 2, 2016
Analysis of JC virus DNA replication using a quantitative and high-throughput assay
Jong Shin1, Paul J Phelan1, Panharith Chhum1
1Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
Abstract:
Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.
Insights
A new assay quantifies JC virus (JCV) DNA replication, crucial for Progressive Multifocal Leukoencephalopathy (PML). This tool aids in understanding JCV replication and developing antiviral drugs for PML.
Area of Science:
- Virology
- Molecular Biology
- Neuroscience
Background:
- Progressive Multifocal Leukoencephalopathy (PML) is a severe neurological disease caused by JC virus (JCV) lytic replication in the central nervous system.
- JC virus (JCV) encodes a large T-antigen (T-ag) helicase essential for viral DNA replication, similar to other polyomaviruses.
Purpose of the Study:
- To develop a quantitative, high-throughput assay for measuring JCV DNA replication.
- To investigate the roles of T-ag domains and viral origin sequences in JCV replication.
- To evaluate the assay's utility in identifying antiviral compounds targeting JCV.
Main Methods:
- Development of a luciferase-based assay using C33A cells, which efficiently accumulate nuclear T-ag for robust JCV replication.
- Utilizing the assay to examine the impact of T-ag domains and origin sequences on viral DNA replication.
- Testing the assay's capacity for antiviral drug screening.
Main Results:
- The developed assay provides a quantitative and high-throughput method for studying JCV DNA replication.
- Studies revealed specific requirements for T-ag domains and origin sequences.
- A significant stimulatory role for the transcription factor NF1 in JCV DNA replication was identified.
- The assay demonstrated effectiveness in inhibitor testing for potential antiviral drug discovery.
Conclusions:
- A novel, robust assay for JCV DNA replication has been established, facilitating further research into PML pathogenesis.
- The findings elucidate critical factors influencing JCV replication, including T-ag domains, origin sequences, and NF1.
- This assay serves as a valuable platform for the discovery of novel antiviral therapeutics against JCV infections.
