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.

Keywords: SARS-CoV-2; protein interactome; protein-protein interactions; two-hybrid analyses.

FIG 1

Bacterial two-hybrid assay used to study the SARS-CoV-2 interactome. (A) (Top) Schematic depiction of the employed transcription-based bacterial two-hybrid system. Interaction between protein moieties X (purple) and Y (slate blue), which are fused to the N-terminal domain of the α subunit of E. coli RNAP (αNTD) and the λCI protein, respectively, stabilizes the binding of RNAP to test promoter placO_L2–62, thereby activating transcription of the lacZ reporter gene. The test promoter bears the λ operator O_L2 centered at position −62 upstream of the transcription start site. (Bottom) E. coli cell containing genetic elements that are involved in the bacterial two-hybrid system. The chromosomal lacZ locus is deleted, and the test promoter and fused lacZ reporter gene are encoded on an F′ episome. The λCI-Y and αNTD-X fusion proteins are encoded on compatible plasmids and produced under the control of IPTG-inducible promoters. (B) List of all tested SARS-CoV-2 ORFs as predicted by the NCBI reference genome (accession number NC_045512.2). The respective nucleotide range for each ORF based on the NCBI reference sequence is indicated, together with the resulting amino acid sequence length. Except for the spike protein, all ORFs were cloned as full-length genes. For spike, we chose to test the interaction of its ectodomain (aa 16 to 1213) to avoid complications due to its N-terminal signal peptide and C-terminal transmembrane domain.

FIG 2

Detection of protein-protein interactions by the bacterial two-hybrid system. Interaction matrix of all tested ORFs. Positive interactions, regardless of the fusion partner, are indicated with purple squares, and self-interactions are indicated by orange-framed squares. Detailed information about fusion constructs for which positive interactions were identified is given in Fig. S1. To avoid data duplication, only one half of the matrix is utilized, while the other is shaded in gray.

FIG 3

Strong SARS-CoV-2 protein-protein interactions identified by B2H assays. Shown are two-hybrid data for strong interactions (arbitrarily defined as >500 Miller units). The indicated ORFs are fused either to the αNTD (α) or to full-length λCI (CI). For the negative controls, the λCI and α fusion proteins were tested in combination with full-length α and full-length λCI, respectively. The interaction of domain 4 of the RNAP σ⁷⁰ subunit (fused to the αNTD) with the flap domain of the RNAP β subunit (fused to λCI) served as a positive control (pos) (84, 85). Data are the averages for three biological replicates (n = 3), and β-galactosidase activities are given in Miller units. Error bars indicate standard deviations. Values indicated with asterisks are significantly different from the negative-control value. ****, P < 0.0001 (one-way ANOVA with Tukey’s multiple-comparison test).

FIG 4

Selective disruption of protein interfaces for proteins with two interaction partners. (A) Depiction of crystal structure (PDB ID 5NFY [29]) of SARS-CoV-1 Nsp10 (pale cyan) in complex with Nsp14 (pale pink). The zoom-in shows amino acids (sticks) chosen for mutational analysis of Nsp10 (orange, olive, and burgundy) and their corresponding main interaction partners in Nsp14 (pale pink). (B) B2H results showing effects of Nsp10 substitutions on its interactions with Nsp14 and with Nsp16. Amino acid substitutions introduced into Nsp10 are given in the box. (C) Depiction of crystal structure of the SARS-CoV-2 Nsp16-Nsp10 protein complex (PDB ID 6W4H [31]) colored, respectively, in pale yellow and pale cyan. An additional N-terminal Nsp10^7–22 region is included and was obtained from the superimposed Nsp10 structure from PDB ID 5NFY (green). The zoom-in shows amino acids (sticks) chosen for mutational analysis of Nsp16 (orange and burgundy) and their corresponding main interaction partners in Nsp10 (pale cyan). (D) B2H results showing effects of Nsp16 substitutions on its interactions with Nsp10 and with Nsp15. Amino acid substitutions introduced into Nsp16 are given in the box. (B and D) The indicated ORFs are fused either to the αNTD (α) or to full-length λCI. For the negative controls, the λCI and α fusion proteins were tested in combination with full-length α (α ctrl) and full-length λCI (λCI ctrl), respectively. Data are averages for three biological replicates (n = 3), and β-galactosidase activities are given in Miller units. Error bars indicate standard deviations. Values indicated with asterisks are significantly different from the wild-type value. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparison test). Black dashed lines in panels A and C represent hydrogen bonds.

FIG 5

Interaction of spike RBD and ACE2 in an oxidizing E. coli strain. (A) Bacterial two-hybrid assays of (A) spike domains (as listed in Fig. S7) tested against ACE2 in BLS148, (B) indicated spike RBD cysteine mutants tested against ACE2 in BLS148 and B2H, or (C) indicated spike RBD circulating variants tested against ACE2 in BLS148. FL, full-length; NTD, N-terminal domain; RBD, receptor binding domain; CTD, C-terminal domain (with or without transmembrane domain [TMD]); Ecto, ectodomain starting at either aa 13 or 16. (D) Schematic depicting amino acid substitutions present in each of three RBD variants tested. The measured effect of each substitution on ACE2 binding is indicated with a dash (no effect), a downward-pointing arrow (weakened binding) or an upward pointing arrow (strengthened binding). (A to C) Spike domains or RBD mutant variants were fused to the αNTD (α), and ACE2 was fused to full-length λCI. For the negative controls, the λCI and α fusion proteins were tested in combination with full-length α (α ctrl) and full-length λCI (λCI ctrl), respectively. Bar graphs show (A) data for one biological replicate or (B and C) averages for three biological replicates (n = 3), and β-galactosidase activities are given in Miller units. Results depicted in panel C were confirmed in a total of seven independent experiments, results of one of which are shown here. Error bars indicate standard deviations. Values indicated with asterisks are significantly different from the negative-control value. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (two-way ANOVA with Tukey’s multiple-comparison test). Western blot analysis indicated that the spike RBD mutants used in panels B and C are present at intracellular levels comparable to the wild-type RBD, ruling out protein instability as a cause for the observed effects (Fig. S8 and S9).