The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2
Norman R. Watts, Elif Eren , Ira Palmer,Paul L. Huang, Philip Lin Huang,Robert H. Shoemaker,Sylvia Lee-Huang, Paul T. Wingfield
Abstract:
The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity.
Introduction:
The continuing emergence of SARS-CoV-2 variants capable of eluding monoclonal antibody therapies, conventional and mRNA vaccines, and the human immune response to infection, has highlighted the importance of identifying additional means of intervention.
Ribosome-inactivating proteins (RIPs) have repeatedly been shown to inhibit a broad range of plant and animal viruses including double-stranded DNA viruses, positive-sense single-stranded RNA viruses, negative-sense RNA viruses, and retroviruses, and to therefore have therapeutic potential (for reviews see [1–6]; for a critique see [7]). Saporin, an RIP derived from soapwort (Saponaria officinalis) has recently been proposed as a therapeutic for SARS-CoV-2, a positive-sense single-stranded RNA virus [8], as has RTAM-PAP1, a fusion protein of the Ricin A-chain and Pokeweed Antiviral Protein isoform 1, obtained from Ricinus communis and Phytolacca americana, respectively [9].
Materials and methods
Recombinant protein production
MAP30 and variants were all well expressed in E. coli. Wild type protein and the mutants MAP30.D43A, MAP30.Y70A, and MAP30.K171A, K215A were purified as previously described using ion-exchange chromatography and gel filtration (S1 Fig) [10]. For the double mutant, some adjustments in pH of ion-exchange columns were required due to a shift in protein isoelectric point. MAP30 with a C-terminal his-tag (MAP30-hist) was purified using Ni-Sepharose chromatography and gel filtration. MAP30 with a C-terminal Tat cell-penetration peptide (GRKKRRQRRRPQ) required urea extraction from bacterial cell extracts and 4 M urea was included in chromatography steps. The urea was removed by step wise dialysis (3, 2, 1 and 0 M urea) in PBS. Momordin and Momordin-Tat (expressed mostly soluble) were purified using the same procedure as MAP30. All proteins were single peaks on gel filtration columns and were concentrated using Amicon Ultra-4, 10,000 NMWL centrifugal filtration units and sterile filtered using Whatman Anotop 10, 0.1 μm filters prior to use. Soluble recombinant human ACE2 (APN01) was provided by Apeiron Biologics.
Results
Protein production and characterization
MAP30 and variants were all well expressed in E. coli as soluble proteins and purified by conventional chromatography (S1A Fig). Similarly, for the Momordin proteins, although the expression levels were lower. MAP30 is a very soluble monomeric protein as established by analytical ultracentrifugation (S1B Fig) and all other proteins were also judged monomeric based on their gel filtration behavior. MAP30 and Momordin are α-helix and β-sheet containing proteins with similar structures (Fig 1A and 1B) and this is reflected by qualitatively similar far-UV circular dichroic spectra derived from secondary structure (S1C Fig). MAP30 is a stable protein which was required for structure determinations by NMR where protein is incubated at 40–42°C for prolonged time periods.
Discussion
Molecular dynamics simulations of MAP30 and Momordin interaction with ribosomal P-protein peptide
In MAP30, the main residue predicted to engage the peptide is Y164, interacting with F10 in replicate simulations 69–72% of the time (S4A and S4B Fig). This involves a π-π face-to-edge interaction. A second set of (charge) interactions involves K215 interacting with D11 and the peptide C-terminus. A third (polar) interaction is between N234 and the L9 carbonyl of the peptide. A fourth set of (charge) interactions involves R167 and K171 with D2, D3, and D4 at the N-terminal end of the peptide.
In Momordin, the main residue predicted to engage the peptide is Y166, interacting with F10 in replicate simulations 37–94% of the time (S4C and S4D Fig). This involves a π-π face-to-edge interaction. A second set of (charge) interactions involves N218, K219, and K231 with D11 and the peptide C-terminus. A third (polar) interaction occurs between S235 and the L9 carbonyl of the peptide.
Conclusion
RIPs have a long and notable history as promising antiviral agents. To our knowledge, this is the first report that RIPs such as MAP30 and Momordin inhibit SARS-CoV-2. For both proteins, the inhibition and cytotoxicity values, ca. 0.2 and 2 μM, respectively, are in the ranges previously reported for other RIPs and viruses. MAP30 and Momordin consistently have essentially identical inhibitory activities on SARS-CoV-2 despite distinct differences in their structures at both their active-sites and ribosome-binding-regions. It is not clear from the structures how to substantially improve their selectivity indices. Our structural analyses point to sites on the viral genome where these RIPs may also exert their effects.
Acknowledgments
All viral inhibition assays were performed at Southern Research, Birmingham, AL. APN01 (soluble recombinant human ACE2) was provided by Apeiron Biologics.
Citation: Watts NR, Eren E, Palmer I, Huang PL, Huang PL, Shoemaker RH, et al. (2023) The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2. PLoS ONE 18(6): e0286370. https://doi.org/10.1371/journal.pone.0286370
Editor: Israel Silman, Weizmann Institute of Science, ISRAEL
Received: January 23, 2023; Accepted: May 15, 2023; Published: June 29, 2023
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All assay data are available in the Dryad Repository (DOI: 10.5061/dryad.z08kprrj4).
Funding: This research was supported by the Intramural Research Program of the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0286370#abstract0
