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A mammalian cell-based reverse two-hybrid system for functional analysis of 3C viral protease of human enterovirus 71
Jin-Ching Lee1, Shin-Ru Shih, Ten-Yuan Chang
1Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei, Taiwan 115, Republic of China.
Abstract:
Although several cell-based reporter assays have been developed for screening of viral protease inhibitors, most of these assays have a significant limitation in that numerous false positives can be generated for the compounds that are interfering with reporter gene detection due to the cellular viability. To improve, we developed a mammalian cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of human enterovirus 71 (EV71) 3C protease and to validate the cytotoxicity of compounds at the same time. In this system, the GAL4 DNA binding domain (M3) and transactivation domain (VP16) were fused, in-frame, with 3C or 3C(mut). The 3C(mut) was an inactivated protease with mutations at the predicted catalytic triad. The reporter plasmid contains a secreted alkaline phosphatase (SEAP) gene under the control of GAL4 activating sequences. We demonstrated that M3-3C-VP16 failed to turn on the expression of SEAP due to the separation of M3 and the VP16 domains by self-cleavage of 3C. In contrast, SEAP expression was induced by the M3-3C(mut)-VP16 fusion protein or the M3-3C-VP16 in cells treated with AG7088, a potent inhibitor of human rhinoviruses (HRVs) 3C protease. Potentially, this protease detection system should greatly facilitate anti-EV71 drug discovery through a high-throughput screening.
Insights
This study introduces a new mammalian cell-based assay to screen for human enterovirus 71 (EV71) protease inhibitors. The assay simultaneously monitors viral protease activity and compound cytotoxicity, reducing false positives in drug discovery.
Area of Science:
- Virology
- Biochemistry
- Drug Discovery
Background:
- Existing cell-based assays for viral protease inhibitors often yield false positives due to cytotoxicity.
- A need exists for improved screening methods that accurately assess compound efficacy and safety.
Purpose of the Study:
- To develop a novel mammalian cell-based assay for monitoring human enterovirus 71 (EV71) 3C protease activity.
- To simultaneously validate compound cytotoxicity, thereby minimizing false positives in antiviral drug screening.
Main Methods:
- A reverse two-hybrid system was employed, fusing GAL4 DNA binding domain (M3) and VP16 transactivation domain with active (3C) or mutated (3C(mut)) EV71 3C protease.
- Reporter gene expression (secreted alkaline phosphatase - SEAP) was driven by GAL4 activating sequences.
- Assay performance was evaluated using the 3C(mut) fusion protein and in cells treated with the human rhinovirus (HRV) 3C protease inhibitor AG7088.
Main Results:
- The M3-3C-VP16 fusion protein failed to induce SEAP expression due to 3C protease self-cleavage.
- SEAP expression was successfully induced by the M3-3C(mut)-VP16 fusion protein.
- Treatment with AG7088 also led to SEAP induction, confirming assay sensitivity to protease inhibition.
Conclusions:
- The developed assay effectively monitors EV71 3C protease activity and assesses compound cytotoxicity concurrently.
- This system offers a robust platform for high-throughput screening, significantly advancing anti-EV71 drug discovery efforts.
