A Bacterial Cell-Based Assay To Study SARS-CoV-2 Protein-Protein Interactions

Area of Science:

• Virology and Molecular Biology
• Protein-protein interactions
• Drug discovery

Background:

• Understanding viral protein interactions is crucial for basic viral biology and developing therapeutics.
• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions are key to addressing the COVID-19 pandemic.
• Existing therapeutics for COVID-19 are limited, highlighting the need for novel approaches.

Purpose of the Study:

• To develop and utilize a bacterial cell-based two-hybrid (B2H) system for analyzing the SARS-CoV-2 proteome.
• To identify and genetically dissect intraviral protein-protein interactions (PPIs) within SARS-CoV-2.
• To investigate the interaction between the SARS-CoV-2 spike protein receptor-binding domain (RBD) and ACE2, and the impact of mutations.

Main Methods:

• Employed a bacterial cell-based two-hybrid (B2H) system to screen the SARS-CoV-2 proteome for protein interactions.
• Developed a modified B2H system to detect disulfide bond-dependent PPIs in a reducing bacterial cytoplasm.
• Analyzed the effect of specific amino acid substitutions in the RBD on its interaction with ACE2.

Main Results:

• Identified 16 distinct intraviral PPIs involving 16 SARS-CoV-2 proteins, with many proteins interacting with multiple partners.
• Successfully detected the interaction between the SARS-CoV-2 RBD and human ACE2 using the modified B2H system.
• Demonstrated that specific RBD mutations found in circulating SARS-CoV-2 variants can perturb the RBD-ACE2 interaction.

Conclusions:

• The B2H system is a versatile and genetically tractable tool for probing viral protein interactions and dissecting their functional significance.
• The findings provide insights into SARS-CoV-2 biology and offer a platform for identifying potential therapeutic targets.
• The modified B2H system's ability to detect disulfide bond-dependent interactions extends its utility to eukaryotic protein studies.