Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms
- Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, United States
- Novartis Institutes for Biomedical Research, United States
Figures
Figure 1 with 1 supplement
Experimental strategy to measure the function of all individual mutations of main protease (Mpro).
(A) Fluorescence resonance energy transfer (FRET)-based reporter screen. The Mpro variants were sorted based on their ability to cleave at the Mpro cut-site, separating the YFP-CFP FRET pair. Cells …
Figure 1—figure supplement 1
Main protease (Mpro) expression in cells harboring the LexA-UbMpro plasmid construct.
(A) Yeast cells transformed with a plasmid expressing C145A Ub-Mpro-his6 under the LexA promoter were grown to exponential phase followed by the addition of 2 µM β-estradiol to induce expression for …
Figure 2 with 1 supplement
Main protease (Mpro) functional scores are reproducible, and variants can be clearly distinguished based on function.
(A) Correlation between biological replicates of functional scores of all Mpro variants for each screen. Red line indicates best fit. (B) Distribution of functional scores for all variants (gray), …
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Figure 2—source data 1
Sequencing counts and functional scores for each amino acid of main protease (Mpro) in both replicates of all three screens.
For each dataset, the sequencing counts, unnormalized functional scores, and normalized functional scores (normalized to average stop = 0, average wild-type barcode = 1) are reported. For the growth screens, the selection coefficients are also reported. All figures in this paper use the data from replicate 1 of each screen.
- https://cdn.elifesciences.org/articles/77433/elife-77433-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Cumulative frequency distributions for all variants (gray), stops (red), and wild-type (WT) barcodes (blue) for all three screens.
Figure 3 with 2 supplements
Heatmap representation of the main protease (Mpro) functional scores measured in the fluorescence resonance energy transfer (FRET) screen (replicate 1).
Arrows represent positions that form β-sheets, coils represent α-helices, and red triangles indicate the catalytic dyad residues H41 and C145.
Figure 3—figure supplement 1
Heatmap representation of scores from the transcription factor (TF) screen (replicate 1).
Arrows represent positions that form beta sheets, coils represent α-helices, and red triangles indicate the catalytic dyad residues H41 and C145.
Figure 3—figure supplement 2
Heatmap representation of scores from the growth screen (replicate 1).
Arrows represent positions that form beta sheets, coils represent α-helices, and red triangles indicate the catalytic dyad residues H41 and C145.
Figure 4
Functional scores reflect fundamental biophysical constraints of main protease (Mpro).
(A) Heatmap representation of the average functional score at each position (excluding stops) in replicate 1 of each screen (see Figure 4—source data 1). (B) The average functional score at each …
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Figure 4—source data 1
Average functional score (excluding stops) at each position of main protease (Mpro) in replicate 1 of each screen.
- https://cdn.elifesciences.org/articles/77433/elife-77433-fig4-data1-v2.xlsx
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Figure 4—source data 2
Comparison of previously measured relative catalytic rates of individual mutations to functional scores.
- https://cdn.elifesciences.org/articles/77433/elife-77433-fig4-data2-v2.docx
Figure 5 with 1 supplement
Functional scores indicate that natural amino acid variants of main protease (Mpro) are generally fit.
(A) Comparison of functional scores in the FRET screen (left panel) and growth screen (right panel) to the number of observations among clinical samples. All missense mutations excluding stops are …
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Figure 5—source data 1
Frequency at which the clinical variants of the main protease (Mpro) gene have been observed.
- https://cdn.elifesciences.org/articles/77433/elife-77433-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Functional scores indicate that natural amino acid variants of main protease (Mpro) are generally fit.
(A) Comparison of functional scores in the transcription factor (TF) screen to the number of observations among clinical samples. All missense mutations excluding stops are indicated with black …
Figure 6 with 1 supplement
Structural distribution of main protease (Mpro) positions that are intolerant to mutation.
(A) Mpro positions that are intolerant of mutations with 17 or more substitutions having null-like function are represented by red spheres on chain A (shown in gray) and pink spheres on chain B …
Figure 6—figure supplement 1
Comparison of the average transcription factor (TF) functional score of each position to conservation observed in a broad sample of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro) homologs.
Figure 7 with 1 supplement
Substrate and inhibitor binding sites are variably sensitive to mutation.
(A) All main protease (Mpro) positions that contact the Nsp4/5 substrate peptide are represented in spheres and colored by their average fluorescence resonance energy transfer (FRET) functional …
Figure 7—figure supplement 1
Substrate and inhibitor binding sites are variably sensitive to mutation.
(A) All main protease (Mpro) positions that contact the Nsp4/5 substrate peptide are represented in spheres and colored by their average transcription factor (TF) functional score (PDB 7T70). The …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (SARS-CoV-2) | ORF1ab/ nsp5A-B | NIH GenBank | NC_045512 | Mpro |
| Strain, Strain background (Saccharomyces cerevisiae) | W303 | Saccharomyces Genome Database | GenBank JRIU00000000 | |
| Antibody | anti-his tag HRP-labelled (Mouse monoclonal) | R&D systems | CAT#: MAB050H | WB (1:4000) |
| Recombinant DNA reagent | Barcoded UbMpro plasmid library | This paper | p416LexA- UbMpro(lib)-N18 | See Materials and Methods section “Generating mutant libraries” |
| Recombinant DNA reagent | Barcoded WT UbMpro plasmid | This paper | p416LexA- UbMpro(WT)-N18 | See Materials and Methods section “Construction of WT Ub-Mpro vector” |
| Recombinant DNA reagent | C145A-Mpro-his6 plasmid | This paper | p416LexA-UbMpro (C145A)-his | See Materials and Methods section “Analysis of Mpro expression” |
| Recombinant DNA reagent | pCyPet-His | Addgene | #14,040 | |
| Recombinant DNA reagent | pYPet-His | Addgene | #14,031 | |
| Recombinant DNA reagent | CyPet-MproCS-YPet fusion gene | This paper | See Materials and Methods section “Generating FRET strain” | |
| Recombinant DNA reagent | pDK-ATC | PMID:28660202 | Integrative bidirectional plasmid with TEF and CUP promoters | |
| Recombinant DNA reagent | pDK-ATG | PMID:28660202 | Integrative bidirectional plasmid with TEF and GPD promoters | |
| Recombinant DNA reagent | DBD-MproCS-AD fusion gene | This paper | See Materials and Methods section “Generating split TF strain” | |
| Commercial assay or kit | KAPA SYBR FAST qPCR Master Mix | Kapa Biosystems | KK4600 | |
| Commercial assay or kit | BCA protein assay kit | Pierce | CAT# 23,225 | |
| Chemical compound, drug | β-Estradiol | Sigma Aldrich | E2768 | |
| Software, algorithm | Scripts to tabulate variant counts | This paper | https://github.com/JuliaFlynn/BolonLab, (copy archived at swh:1:rev:b54d80818c2681fb89533ae330c18a3d39f32ab6) | See Materials and Methods section “Analysis of Illumina sequencing data” |
| Software, algorithm | Scripts to associate barcodes with variants | This paper | https://github.com/JuliaFlynn/PacBio_barcode_assocation, (copy archived at swh:1:rev:29eac92475a9ff8e24fb390986c865b504c03f51) | See Materials and Methods section “Barcode Association” |
| Software, algorithm | GraphPad Prism 9 | Graphpad.com | RRID:SCR_008520 | |
| Software, algorithm | Flowjo v.10.8.0 | BD Biosciences | RRID:SCR_008520 | |
| Software, algorithm | Pymol v. 2.5.2 | Schrödinger | RRID:SCR_000305 | |
| Software, algorithm | MatPlotLib | http://matplotlib.sourceforge.net | RRID:SCR_008624 | |
| Sequence-based reagent | Sequencing primers | This paper | See Supplementary file 1 | |
| Sequence-based reagent | Site-directed mutagenesis primers | This paper | See Supplementary file 1 |
Additional files
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Supplementary file 1
List of oligomers used in this study.
- https://cdn.elifesciences.org/articles/77433/elife-77433-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/77433/elife-77433-transrepform1-v2.docx
