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Updated: Apr 16, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against
Zachary D Stolp1, Cameron A Smurthwaite1, Connor Reed1
1Department of Biology, San Diego State University, San Diego, CA, USA.
Abstract:
The DenV pre-membrane protein (prM) is a crucial chaperone for the viral envelope protein, preventing premature fusion with vesicles during viral export. prM molecules in immature particles are cleaved by host proteases, leading to mature fusogenic virions. Blockade of prM cleavage would restrict fusion and represents a novel druggable opportunity against DenV. We have thus established a cell-based platform to monitor prM processing that relies on an engineered two-tag scaffold that travels to the cell surface through the secretory pathway. The assay discriminates between a single cell-surface tag when prM is cleaved and two tags when it is not, as detected through fluorescent-coupled antibodies by flow cytometry. The assay, miniaturized into a 96-well plate format, was multiplexed with the HIV-1 envelope boundary, also cleaved in the same pathway. A pilot screen against 1280 compounds was executed, leading to the identification of a potential active and corroborating the robustness of our assay for large-scale screening. We describe for the first time a cell-based assay that monitors DenV prM processing within the classical secretory pathway, which was exploited to identify a potential novel drug against DenV.
Insights
Researchers developed a novel cell-based assay to track Dengue virus (DENV) prM protein processing. This assay identified a potential new drug candidate to inhibit DENV fusion and replication.
Area of Science:
- Virology and Molecular Biology
- Drug Discovery and Development
Background:
- The Dengue virus pre-membrane protein (prM) is essential for viral maturation, controlling fusion events during viral export.
- Cleavage of prM by host proteases is critical for generating infectious virions; blocking this cleavage is a potential antiviral strategy.
- Current methods lack efficient cell-based assays to monitor prM processing for drug screening.
Purpose of the Study:
- To establish and validate a novel cell-based assay for monitoring Dengue virus prM protein processing.
- To utilize this assay for identifying novel compounds that inhibit prM cleavage and thus viral fusion.
- To demonstrate the assay's utility in a pilot drug screening campaign.
Main Methods:
- Development of a cell-based platform using an engineered two-tag scaffold that reports prM cleavage status on the cell surface.
- Detection of prM processing via flow cytometry using fluorescent-coupled antibodies to distinguish between single-tag (cleaved) and two-tag (uncleaved) states.
- Miniaturization of the assay to a 96-well format and multiplexing with HIV-1 envelope processing for broader applicability.
Main Results:
- The developed assay accurately monitors prM processing in real-time within the secretory pathway.
- A pilot screen of 1280 compounds identified a potential active compound that interferes with prM cleavage.
- The assay demonstrated robustness and suitability for large-scale drug screening against Dengue virus.
Conclusions:
- A novel, robust cell-based assay for monitoring Dengue virus prM processing has been successfully developed.
- This platform enables the identification of compounds targeting prM cleavage, representing a new avenue for antiviral drug discovery.
- The findings support the development of novel therapeutics to combat Dengue virus infections by inhibiting viral fusion.
