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

  1. Benjamin L. Springstein
  2. Padraig Deighan
  3. Grzegorz Grabe
  4. Ann Hochschild

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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 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.

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 bacteria-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current pandemic. We identify sixteen distinct intraviral protein-protein interactions, involving sixteen 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 bacteria-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 investigate 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.

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    SciScore for 10.1101/2021.10.07.463611: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Upon gel separation, proteins were transferred to Amersham Protran 0.45 NC nitrocellulose membranes (Cytiva, MA, USA) using a Trans-Blot Turbo Transfer System (BioRad Hercules, CA, USA), blocked in blocking buffer (TBST: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.1 % Tween-20 supplemented with 5% non-fat dry milk) for 30 min at RT and then incubated with mouse anti-a-NTD and rabbit anti-CI primary antibodies (both 1:3,000 dil.
    anti-a-NTD
    suggested: None
    anti-CI
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    FW102 OL2-62 and BLS148 strains were used for bacterial two-hybrid assays.
    OL2-62
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Finally, the β-galactosidase reporter present on the F’ was introduced into BLS133 by mating with strain FW102 OL2-62 [84].
    OL2-62
    suggested: None
    Recombinant DNA
    SentencesResources
    Next, TSS competent BLS128 cells were created according to [83], transformed with pCP20 (encoding the yeast Flp recombinase gene to flip out the kanamycin resistance gene) and grown over night at 30 °C on LB plates containing carbenicillin (100 μg/ml; Carb100).
    pCP20
    suggested: None
    Except for spike, Nsp2, Nsp3, RNA-Polymerase (Nsp12) and helicase (Nsp13), all full-length codon-optimized gene fragments were digested with NotI+BamHI, purified by DNA Clean & Concentrator kit (Zymo Research) and then ligated into 50 ng NotI+BamHI-digested pBRα or pACλCI using T4 ligase (NEB) according to standard protocols generating the plasmids listed in Supplementary Table 1A. Spike: For pS63, the spike full-length (FL) sequence was amplified from E. coli codon-optimized gene fragments by SARS_67+SARS_68 and cloned into NotI+BamHI-digested pBRα by Gibson assembly.
    pS63
    suggested: None
    pBRα
    suggested: RRID:Addgene_53731)
    For pS64, the NTD sequence was amplified from pS63 by SARS_67+SARS_69 and then cloned into NotI+BamHI-digested pBRα by Gibson assembly.
    pS64
    suggested: None
    For pS66, the CTD sequence was amplified from pS63 by SARS_68+SARS_72 and then cloned into NotI+BamHI-digested pBRα by Gibson assembly.
    pS66
    suggested: None
    For pS67, the Ectodomain (aa 13-1213) was amplified from pS63 by SARS_73+SARS_74 and then cloned into NotI+BamHI-digested pBRα by Gibson assembly.
    pS67
    suggested: None
    For pS68, the Ectodomain (aa 16-1213) was amplified from pS63 by SARS_74+SARS_75 and then cloned into NotI+BamHI-digested pBRα by Gibson assembly.
    pS68
    suggested: None
    pS70 was generated by site-directed mutagenesis (SDM; see below) using primers SARS_17+SARS_76 and pS63 as a template.
    pS70
    suggested: None
    For pS72, FL spike was amplified from pS63 by SARS_77+SARS_78 and then cloned into NotI+BamHI-digested pACλCI by Gibson assembly.
    pS72
    suggested: None
    For pS73, the NTD sequence was amplified from pS63 by SARS_77+SARS_79 and then cloned into NotI+BamHI-digested pACλCI by Gibson assembly.
    pS73
    suggested: None
    For pS74, the RBD sequence was amplified from pS63 by SARS_80+SARS_81 and then cloned into NotI+BamHI-digested pACλCI by Gibson assembly.
    pS74
    suggested: None
    For pS75, the CTD sequence was amplified from pS63 by SARS_78+SARS-82 and then cloned into NotI+BamHI-digested pACλCI by Gibson assembly.
    pS75
    suggested: None
    For pS76, the Ectodomain (aa 131213) was amplified from pS63 by SARS_83+SARS_84 and then cloned into NotI+BamHI-digested pACλCI by Gibson assembly.
    pS76
    suggested: None
    For pS77, the Ectodomain (aa 16-1213) was amplified from pS63 by SARS_84+SARS_85 and then cloned into NotI+BamHI-digested pACλCI by Gibson assembly.
    pS77
    suggested: None
    pACλCI
    suggested: RRID:Addgene_53730)
    pS79 was generated by SDM using primers SARS_17+SARS_86 and pS63 as a template.
    pS79
    suggested: None
    Nsp2: The Nsp2 sequence was ordered as two single gene fragments, which were further amplified by PCR using primers SARS_109+SARS_110 or SARS_111+SARS_112 and then cloned into NotI+BamHI-digested pBRωGP by Gibson assembly, creating pS85.
    pS85
    suggested: None
    Next, the whole Nsp2 open reading frame (ORF) was cut from pS85 by NotI+BamHI and inserted into 50 ng NotI+BamHI-digested pBRa or pACλCI using T4 ligase (NEB) according to standard protocols, creating pS179 and pS180, respectively.
    pBRa
    suggested: None
    pS179
    suggested: None
    pS180
    suggested: None
    Next, the whole Nsp3 open reading frame (ORF) was cut from pS89 by NotI+BamHI and inserted into 50 ng NotI+BamHI-digested pBRa or pACλCI using T4 ligase (NEB) according to standard protocols, creating pS181 and pS182, respectively.
    pS89
    suggested: None
    pS181
    suggested: None
    RNA-Polymerase (Nsp12): The RNA-Polymerase sequence was ordered as three single gene fragments, which were further amplified by PCR using primers SARS_131+SARS_132, SARS_133+SARS_134 or SARS_135+SARS_136 and then cloned into NotI+BamHI-digested pBRωGP by Gibson assembly, creating pS173.
    pBRωGP
    suggested: None
    pS173
    suggested: None
    Next, the whole Nsp3 open reading frame (ORF) was cut from pS89 by NotI+BamHI and inserted into 50 ng NotI+BamHI-digested pBRa or pACλCI using T4 ligase (NEB) according to standard protocols, creating pS181 and pS182, respectively. helicase (Nsp13): The helicase sequence was ordered as two single gene fragments, which were further amplified by PCR using primers SARS_125+SARS_126 or SARS_127+SARS_128 and then cloned into NotI+BamHI-digested pBRωGP by Gibson assembly, creating pS169.
    pS182
    suggested: None
    pS169
    suggested: None
    Next, the whole Nsp2 open reading frame (ORF) was cut from pS85 by NotI+BamHI and inserted into 50 ng NotI+BamHI-digested pBRa or pACλCI using T4 ligase (NEB) according to standard protocols, creating pS221 and pS222, respectively.
    pS221
    suggested: None
    pS222
    suggested: None
    ACE2: The ACE2 N-terminal peptidase domain (aa 19-615) was ordered as a single gene fragment and then further amplified by PCR using primers SARS_264+SARS_265 or SARS_266+SARS_267 and then cloned into 50 ng NotI+BamHI-digested pBRα or pACλCI by Gibson assembly, creating pS260 and pS261, respectively.
    pS260
    suggested: None
    pS261
    suggested: None
    The annealed oligos were diluted 1:50 and then ligated into 50 ng NotI+BamHI-digested pBRα or pACλCI using T4 ligase (NEB), generating pS197 and pS198, respectively.
    pS197
    suggested: None
    pS198
    suggested: None
    pS254 was generated by SDM using primers SARS_253+SARS_254 and plasmid pS215 as a template.
    pS254
    suggested: None
    pS256 was generated by SDM using primers SARS_256+SARS_257 and plasmid pS215 as a template.
    pS256
    suggested: None
    pS215
    suggested: None
    pS257 was generated by SDM using primers SARS_256+SARS_257 and plasmid pS254 as a template.
    pS257
    suggested: None
    pS262 was generated by Gibson assembly using primers SARS_268+SARS_269 and plasmid pS196 as a template.
    pS262
    suggested: None
    pS263 was generated by Gibson assembly using primers SARS_270+SARS_271 and plasmid pS196 as a template.
    pS263
    suggested: None
    pS196
    suggested: None
    pS264 was generated by Gibson assembly using primers SARS_272+SARS_273 and plasmid pS196 as a template.
    pS264
    suggested: None
    pS267 was generated by Gibson assembly using primers SARS_280+SARS_281 and plasmid pS65 as a template.
    pS65
    suggested: None
    pS271 was generated by Gibson assembly using primers SARS_287+SARS_288 and plasmid pS65 as a template.
    pS271
    suggested: None
    pS272 was generated by Gibson assembly using primers SARS_289+SARS_290 and plasmid pS65 as a template.
    pS272
    suggested: None
    pS273 was generated by Gibson assembly using primers SARS_289+SARS_290 and plasmid pS271 as a template.
    pS273
    suggested: None
    pS275 was generated by Gibson assembly using primers SARS_295+SARS_296 and plasmid pS65 as a template.
    pS275
    suggested: None
    pS276 was generated by Gibson assembly using primers SARS_291+SARS_292 and plasmid pS65 as a template.
    pS276
    suggested: None
    pS277 was generated by Gibson assembly using primers SARS_293+SARS_294 and plasmid pS65 as a template.
    pS277
    suggested: None
    pS279 was generated by Gibson assembly using primers SARS_293+SARS_294 and plasmid pS267 as a template.
    pS279
    suggested: None
    pS267
    suggested: None
    pS280 was generated by Gibson assembly using primers SARS_293+SARS_294 and plasmid pS278 as a template. β-galactosidase assays: β-galactosidase assays to study the SARS-CoV-2 interactome were performed essentially as described previously [85].
    pS280
    suggested: None
    pS278
    suggested: None
    Software and Algorithms
    SentencesResources
    Plasmid sequence integrity was verified by Sanger sequencing from Genewiz or Quintarabio (both Boston, MA, USA).
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Structural images were prepared using PyMOL software (Schrodinger, LLC. 2010.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Statistical analysis: Presentation of bacterial two-hybrid data and statistical analysis using one-way or two-way ANOVA with Tukey’s or Dunnett’s multiple comparison test was done using GraphPad Prism (v. 9.1.2; San Diego, CA, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    These discrepancies highlight the importance of employing multiple assay systems, each with its own inherent limitations, to maximize the likelihood of obtaining a complete picture. Because of its experimental accessibility, we expect that our B2H assay will be useful in evaluating other viral proteomes, particularly by taking advantage of our oxidizing reporter strain that better approximates the eukaryotic cell environment in allowing for proper disulfide bond formation in the E. coli cytoplasm [51,52]. To further extend the spectrum of testable viral and eukaryotic PPIs, the system could be augmented to enable the detection of phosphorylation-dependent PPIs by introducing specific mammalian kinases into our reporter stain [53]. Given that many mammalian (and presumably viral) proteins are constitutively phosphorylated in yeast [54–56], a lack of properly phosphorylated proteins in our B2H system could explain, at least in principle, why some SARS-CoV-2 PPIs were identified only in the Y2H screens [6] and not in our system (Supplementary Fig. 3). We note, however, that a comprehensive phosphoproteomics analysis of SARS-CoV-2-infected cells [57] suggests that other than interactions involving the N protein, which was found to be phosphorylated at multiple sites, most of the viral PPIs that were detected by Y2H analysis but not in our B2H system involve proteins that were not detectably phosphorylated. Genetic dissection of specific SARS-CoV-2 PPIs: A benefit of two-hybrid ap...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2021.10.07.463611v1 on bioRxiv