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Updated: Sep 16, 2025
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Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods
Published on: December 21, 2019
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Subcellular determinants of orthoflavivirus protease activity
Lochlain Corliss1, Chad M Petit2, Nicholas J Lennemann1
1Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
The Journal of Biological Chemistry
|July 6, 2025
Summary
Orthoflavivirus proteases are targeted for new antivirals. This study reveals endoplasmic reticulum localization and membrane proximity are key for viral protease cleavage, impacting viral fitness.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Orthoflaviviruses cause millions of infections annually with no specific antiviral treatments.
- Viral proteases are essential for orthoflavivirus replication and are promising therapeutic targets.
- The molecular mechanisms governing intracellular substrate cleavage by orthoflavivirus proteases remain poorly understood.
Purpose of the Study:
- To investigate the subcellular determinants of orthoflavivirus protease cleavage.
- To identify molecular factors influencing substrate recognition and processing by viral proteases.
- To understand how protease cleavage efficiency impacts viral fitness and infection.
Main Methods:
- Development and utilization of a fluorescent protease-activity reporter system.
- Modification of the reporter platform to probe subcellular localization and membrane proximity effects.
- Alteration of substrate recognition motifs to mimic native viral polyprotein cleavage sites.
- Live-cell imaging to monitor cleavage kinetics.
- Analysis of infectious clones with modified cleavage sequences.
Main Results:
- Endoplasmic reticulum (ER) subdomain localization and membrane proximity of cleavage sites were identified as novel determinants for protease activity.
- The protease exhibited inefficient processing of the NS4A|2K polyprotein junction sequence.
- Cleavage at the NS4A|2K motif was significantly delayed compared to other sites.
- Introduction of a more efficient cleavage sequence into the NS4A|2K junction abolished viral recovery.
Conclusions:
- Substrate cleavage site localization within specific ER subdomains and proximity to membranes are critical molecular determinants for orthoflavivirus protease function.
- Sequence specificity at the NS4A|2K junction plays a vital role in polyprotein processing and is essential for productive orthoflavivirus infection.
- Understanding these determinants provides insights for developing targeted antiviral therapies against orthoflaviviruses.
Keywords:
endoplasmic reticulumflaviviruslive-cell imagingmolecular toolsorthoflaviviruspolyproteinpositive-sense RNA virusproteaseMore Related Videos
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