A new bacterial two-hybrid assay to study SARS-CoV-2 interactome

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This virus is a zoonotic pathogen belonging to the family Coronaviridae of the genus betacoronaviruses. It is around 30kb in size and is an extremely infectious single-stranded and positive-sense RNA virus.

The Functional Roles of SARS-CoV-2 Proteins             

SARS-CoV-2 encodes structural (spike, envelope, membrane, and nucleocapsid) and non-structural (Nsp1-Nsp16) proteins. The non-structural proteins are transcribed from two major open reading frames (ORF1a and ORF1b), which produce Nsps. It also contains around six accessory proteins, i.e., ORF3a, ORF6, 60 ORF7a, ORF7b, ORF8, and ORF10.

The primary function of the spike (S) protein of SARS-CoV-2 is to establish the viral infection. The S protein consists of two functionally distinct subunits, S1 and S2. The receptor-binding domain (RBD), present in the S1 domain, interacts with the host cell's angiotensin-converting enzyme 2 (ACE2) and the S2 domain fuses the cell membranes. In this way, the virus gains entry into the host cell. The primary function of the Nsps is initiating viral replication and, thus, promoting viral RNA synthesis by the RNA-dependent RNA polymerase Nsp12.

Both structural proteins, as well as Nsps, take part in immune evasion. In the case of the Nsps, it suppresses interferon response and, thereby, aids in the host's innate immune system evasion. Accessory proteins are non-conserved and are highly variable among different coronavirus species. Although functional roles of these proteins are mainly unknown, they are known to take part in the evasion of immune responses and disease severity.

SARS-CoV-2 Proteins and Development of Therapeutics

To date, SARS-CoV-2 has claimed more than 4.85 million lives worldwide. Therefore, to protect individuals from this virus, there is a continuing need for more effective therapeutics. To develop SARS-CoV-2 therapeutics, understanding the intraviral and viral-host protein-protein interactions (PPIs) associated with COVID-19 infection is essential.

Previous studies have shown that Nsp10 and Nsp8 are potential inducers of the "cytokine storm". Cytokine storm is a dysregulated and hyperactive immune response that occurs in severe COVID-19 disease and often causes death. These studies have indicated that Nsp8 and Nsp10 could be efficacious targets for drug development. In addition to its role in viral infection, RBD is also a critical determinant of viral tropism, which is a major target for SARS-CoV-2-neutralizing antibodies.

Mutations have led to the emergence of several SARS-CoV-2 variants which have been classified as variants of interest (VOI) and variants of concern (VOC). All the variants have shown mutations in the S protein, including in the RBD region. The mutation rate of SARS-CoV-2 is found to be relatively low when compared with other RNA viruses. In comparison to the original strain, SARS-CoV-2 VOCs have shown increased transmissibility, high virulence, and a decrease in the effectiveness of available vaccines and diagnostics.

A New Bacterial Two-hybrid (B2H) System

The development of novel methods for identifying and dissection of the interactions of virally encoded proteins is essential for understanding basic viral biology. Moreover, as stated above, these also provide a foundation for therapeutic advances. Recently, scientists have developed a bacterial two-hybrid (B2H) system to study the PPIs of SARS-CoV-2 in a heterologous non-eukaryotic system. This study is available on the bioRxiv* preprint server. Put simply, the B2H system could be used to analyze the SARS-CoV-2 proteome.

In this study, sixteen distinct intraviral protein-protein interactions (PPIs) were identified, involving sixteen proteins, and many of the recently identified proteins were found to interact with more than one partner. Researchers also carried out genetic dissection of these interactions via their model and were able to identify selectively disruptive mutations. Thus, this system aids the genetic dissection of protein interactions and discovers their functional roles.

Researchers demonstrated that a modified B2H system could detect disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm. Additionally, the spike RBD-ACE2 interaction was studied, and the effect of mutations found in VOCs was investigated using this system. Researchers found that the RBD-ACE2 interaction got perturbed by several RBD amino acid substitutions in circulating VOCs. Thus, in principle, this system could also facilitate the identification of potential therapeutics targeting the interactions of virally encoded proteins.

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 placOL2-62, thereby activating transcription of the lacZ reporter gene. The test promoter bears the λ operator OL2 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 #: 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 16-1213) to avoid complications due to its N-terminal signal peptide and C-terminal transmembrane domain.
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 placOL2-62, thereby activating transcription of the lacZ reporter gene. The test promoter bears the λ operator OL2 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 #: 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 16-1213) to avoid complications due to its N-terminal signal peptide and C-terminal transmembrane domain.

Importance of the Study

The study highlights the use of the bacteria-based system for studying the interactions of the proteins encoded by SARS-CoV-2. For the study of viral PPIs, B2H is a convenient and economical genetic tool. According to the authors, this is the first bacteria-based viral interactome that describes sixteen different intraviral PPIs from SARS-CoV-2. In addition, one of the advantages of using a non-eukaryotic system (the B2H assay) is the lack of bridging factors that typically complicate the interpretation of positive results.

Selective disruption of protein interfaces for protein 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). 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. Additional N-terminal Nsp107-22 region is included and was obtained from superimposed Nsp10 structure from PDB ID: 5NFY (green). 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,D) Indicated ORFs are fused either to the αNTD (indicated as α) or to full-length λCI. α and λCI negative controls express full-length α and full length λCI, respectively. Bar graphs show the averages of three biological replicates (n=3) and β-galactosidase activities are given in Miller units. Error bars indicate the standard deviation. Values indicated with asterisks are significantly different from the WT. 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 A and C represent hydrogen bonds.
Selective disruption of protein interfaces for protein 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). 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. Additional N-terminal Nsp107-22 region is included and was obtained from superimposed Nsp10 structure from PDB ID: 5NFY (green). 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,D) Indicated ORFs are fused either to the αNTD (indicated as α) or to full-length λCI. α and λCI negative controls express full-length α and full length λCI, respectively. Bar graphs show the averages of three biological replicates (n=3) and β-galactosidase activities are given in Miller units. Error bars indicate the standard deviation. Values indicated with asterisks are significantly different from the WT. 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 A and C represent hydrogen bonds.

A limitation of this bacterial system is the lack of machinery for promoting potentially post-translational modifications, e.g., protein phosphorylation and protein glycosylation.

The current study revealed that the newly developed oxidizing B2H reporter strain was able to detect the SARS-CoV-2 spike RBD-ACE2 interaction. Furthermore, it could also characterize the effects of several RBD substitutions present in circulating variants.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Priyom Bose

Written by

Dr. Priyom Bose

Priyom holds a Ph.D. in Plant Biology and Biotechnology from the University of Madras, India. She is an active researcher and an experienced science writer. Priyom has also co-authored several original research articles that have been published in reputed peer-reviewed journals. She is also an avid reader and an amateur photographer.

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