Mutational analysis of SARS-CoV-2 main protease

A recent study posted to the bioRxiv* preprint server discussed the findings of a comprehensive mutational analysis of the main protease (Mpro) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Study: Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms. Image Credit: Kateryna Kon/ShutterstockStudy: Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms. Image Credit: Kateryna Kon/Shutterstock

The coronavirus disease 2019 (COVID-19) pandemic has caused unprecedented health and economic crisis across the globe disrupting normal life. Various types of drugs were initially repurposed to treat COVID-19 before the advent of SARS-CoV-2 vaccines. So far, more than eight billion doses of COVID-19 vaccines have been administered. Despite that, infections continue to rise massively across many countries dominated by the recently emerged Omicron variant.

Although several antiviral drugs and monoclonal antibodies against SARS-CoV-2 are used in COVID-19 treatment, the Mpro, also known as 3CL protease, has emerged as a promising drug target with several drugs in the pipeline. Mpro cleaves the two polyproteins (pp1a and pp1ab) of SARS-CoV-2, which are indispensable for the viral life cycle in the host.

The study

In the present study, the authors carried out a comprehensive mutational scan of the SARS-CoV-2 Mpro and analyzed the functions of all point mutations resulting in single amino acid substitutions. The researchers developed three high throughput orthogonal screens in yeast to functionally distinguish Mpro. Mpro is expressed as Ubiquitin (Ub)- Mpro fusion protein and Ub is cleaved subsequently. Mpro expression was inducible by β-estradiol under the tight and precise control of the LexA-ER-AD transcription factor. The bacterial DNA-binding protein LexA, human estrogen receptor (ER), and B112 activation domain make the LexA-ER-AD construct. To control the expression of Ub- Mpro, 4 LexA DNA-binding sequences (LexA boxes) were placed upstream of Ub-Mpro.

In the first screen, the Mpro activity was measured by fluorescence resonance energy transfer (FRET) loss in the Nsp4/5 cut sequence flanked on both sides by fluorescent proteins. The second screening method is the same as the first based on FRET loss, but the transcription factor for green fluorescent protein (GFP) expression is inactivated by the protease activity of Mpro. The final screening is based on the Mpro toxicity to yeast cells decreasing yeast growth. After selecting mutations in the three screens, deep sequencing analysis was performed to quantify the functions based on the enrichment or depletion of each Mpro variant.


In the first screen, the FRET loss was measured by fluorescence-activated single-cell sorting (FACS) due to the Mpro expression in a β-estradiol-dependent manner. An amino acid substitution (C145A) at the catalytic site failed to reproduce the earlier effect of FRET loss indicating that FRET signal change requires a catalytically functional Mpro.

In the second screening assay, the DNA-binding and activation domains of the Gal4 transcription factor flanked the Nsp4/5 cleavage site and the inactivation of a transcription factor by a functional Mpro reduces GFP expression in a β-estradiol-dependent fashion. Mpro is toxic to yeast cells likely due to its protease activity. Yeast growth varies with the concentration of β-estradiol that induces Ub-Mpro and a high Mpro expression is negatively correlated with yeast growth. The authors performed a systematic mutational analysis and integrated the three screening assays to ascertain the functional impact of each point mutation at individual sites in Mpro and each site was replaced with 19 amino acids and a stop codon.

The C145 and H41 mutations at the essential catalytic dyad rendered a functionally inactive Mpro. The team found about 24 sites that had low mutation tolerance and only four of them (C145, H41, H163, and D187) had direct contact with the substrate. The 24 sites were found to be highly conserved across SARS-CoV-2 homologs and sites like G143, H163, D187, and Q192 were highly sensitive to mutations but the sites M49, N142, E166, and Q189 were highly tolerant to mutations. The results revealed that the N142, E166, and Q189 mutations were compatible with Mpro function and proposed that these extremely tolerant sites have a high potential to develop or evolve inhibitor/drug resistance. It was observed that three mutations Q189E, E166A, and E166Q might have potential resistance against the Mpro inhibitor PF-07321332, which is Pfizer's approved drug.


In this study, the team employed yeast screening assays and integrated them with mutational scanning to identify mutations with the functional impact that resulted in the identification of mutation-tolerant and sensitive locations within Mpro. Further, the authors found substitutions that had the potential to induce resistance against the FDA-approved Mpro inhibitor, PF-07321332.

To conclude, the findings of the present study can guide the development of Mpro inhibitor drugs that are less vulnerable to resistance, by promoting drug-protein interactions at mutation-sensitive sites and avoiding mutation-tolerant residues.

While these results are promising, the study was performed on a single Mpro cleavage site out of 11 cleavage sites and more research is required on the other cleavage sites to completely understand the selection pressures on Mpro.

*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:
Tarun Sai Lomte

Written by

Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.


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