A bacteria-based assay to study SARS-CoV-2 protein-protein interactions

Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of COVID-19, an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of SARS-CoV-2-encoded proteins. Here we describe the use of a convenient bacteria-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identify sixteen distinct intraviral protein-protein interactions (PPIs), involving sixteen proteins. We find that many of the identified proteins interact with more than one partner. We further show how our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm and we use this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examine how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins.