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

ABSTRACT: 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 coronavirus disease 2019 (COVID-19), an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of proteins encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we describe the use of a convenient bacterial cell-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identified 16 distinct intraviral protein-protein interactions (PPIs), involving 16 proteins. We found that many of the identified proteins interact with more than one partner. Further, 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 used 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 examined 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. IMPORTANCE Understanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here, we describe the use of a versatile bacterial cell-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current COVID-19 pandemic. We identified 16 distinct intraviral protein-protein interactions, involving 16 proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacterial cell-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically, its receptor-binding domain) and its cognate human cell surface receptor ACE2, and we investigated the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.

SUBMITTER: Springstein BL

PROVIDER: S-EPMC8593686 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionThe COVID-19 pandemic illustrates the need for serology diagnostics with improved accuracy. While conventional serology based on recognition of entire proteins or subunits thereof has made significant contribution to the antibody assessment space, it often suffers from sub-optimal specificity. Epitope-based, high-precision, serology assays hold potential to capture the high specificity and diversity of the immune system, hence circumventing the cross-reactivity with closely related microbial antigens.MethodsWe herein report mapping of linear IgG and IgA antibody epitopes of the SARS-CoV-2 Spike (S) protein in samples from SARS-CoV-2 exposed individuals along with certified SARS-CoV-2 verification plasma samples using peptide arrays.ResultsWe identified 21 distinct linear epitopes. Importantly, we showed that pre-pandemic serum samples contain IgG antibodies reacting to the majority of protein S epitopes, most likely as a result of prior infection with seasonal coronaviruses. Only 4 of the identified SARS-CoV-2 protein S linear epitopes were specific for SARS-CoV-2 infection. These epitopes are located at positions 278-298 and 550-586, just proximal and distal to the RBD, as well as at position 1134-1156 in the HR2 subdomain and at 1248-1271 in the C-terminal subdomain of protein S. To substantiate the applicability of our findings, we tested three of the high-accuracy protein S epitopes in a Luminex assay, using a certified validation plasma sample set from SARS-CoV-2 infected individuals. The Luminex results were well aligned with the peptide array results, and correlated very well with in-house and commercial immune assays for RBD, S1 and S1/S2 domains of protein S.ConclusionWe present a comprehensive mapping of linear B-cell epitopes of SARS-CoV-2 protein S, that identifies peptides suitable for a precision serology assay devoid of cross-reactivity. These results have implications for development of highly specific serology test for exposure to SARS-CoV-2 and other members of the coronaviridae family, as well as for rapid development of serology tests for future emerging pandemic threats.

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

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