A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Infectious diseases, particularly viral infections, pose significant global health challenges due to rapid mutation and host-pathogen interactions.
• Viruses present unique difficulties in treatment development owing to their adaptability and complex molecular mechanisms within host cells.

Purpose of the Study:

• To develop and validate a cell-based multiplexed assay system for monitoring viral proteolytic events.
• To assess the utility of this system in examining viral protease activity and host-mediated viral protein cleavage within their natural subcellular compartments.

Main Methods:

• A cell-based multiplexing strategy was employed, adapting two independent assays to monitor HIV-1 protease activity and host-mediated cleavage of the HIV-1 envelope protein.
• Multiplexing was achieved either by combining distinct cell lines or by engineering single cell lines to express both assays.
• Stable cell expression was utilized to ensure assay robustness and clear discrimination between different proteolytic events.

Main Results:

• The developed multiplexed system successfully monitored specific proteolytic events, including viral protease activity and host protein cleavage of viral components.
• The assays demonstrated robustness and clear discrimination, enabling efficient screening.
• The approach allowed for the examination of proteolysis within the relevant subcellular compartments.

Conclusions:

• Cell-based multiplexed assays are powerful tools for studying viral proteolysis and identifying targets for antiviral drug discovery.
• This approach enhances the capacity to screen for inhibitors of viral infectivity and spread by monitoring critical proteolytic events.
• Monitoring proteolysis in its natural subcellular context is significant for understanding viral pathogenesis and developing effective interventions.