Structural analysis of monkeypox virus to guide the development of comprehensive antiviral agents

Structural analysis of monkeypox virus to guide the development of comprehensive antiviral agents

In a recent study published in bioRxiv * Preprint server: Researchers examined the crystalline structure of monkeypox virus (MPX) (MPXV) and the complex of VP39, a 2′-O-RNA methyltransferase (MTase), and sinefungin, a pan-MTase inhibitor.

Studie: Die Struktur der 2′-O-Ribose-Methyltransferase VP39 des Affenpockenvirus im Komplex mit Sinefungin bildet die Grundlage für das Inhibitordesign. Bildnachweis: Marina Demidiuk/Shutterstock

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources

The number of MPX cases is increasing every hour worldwide and could indicate a new pandemic. Structural analysis of MPXV could be helpful in developing effective antiviral agents to combat MPXV. Poxviruses encode decapping-type enzymes to prevent the accumulation of double-stranded ribonucleic acid (dsRNA) during infection, which could trigger innate antiviral immune responses. MPXV encodes the pox enzyme, which inhibits the cGAS-STING (cyclic GMP-AMP synthase stimulator of interferon genes) pathway triggered by ds-deoxyribonucleic acid (dsDNA).

Methylation of the initial nucleotide (nt) of the mature MPXV cap (or cap-1) at the 2′-O-ribose position was documented. MTase is required by the Poxviridiae family of viruses (including MPXV) for cap-0 synthesis and by adding another methyl group at the 2′-O position of the proximal ribose, the immature cap (cap-0) can be converted into the mature cap. The step is important for preventing the development of innate immune responses and is catalyzed by VP39, the 2′-O MTase of MPXV.

About the study

In the present study, researchers examined the VP39-sinefungin complex structure of MPXV to improve understanding of the mechanisms of inhibition of the VP39 molecule by sinefungin. They also compared the structure to 2′-O-MTases from single-stranded RNA viruses (ssRNA) such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The VP39 gene of the MPXV USA-May22 strain was codon optimized for expression in E. coli for subsequent synthesis and cloning. E. coli BL21 cells were transformed with VP39-expressing plasmid and IPTG (isopropyl-bD-thiogalactopyranoside) was added, followed by purification of the recombinant VP39. The cells were centrifuged, lysed, and the lysate was subjected to chromatographic analysis. VP39 was concentrated and mixed with sinefungin for crystallization-based experiments.

The initially formed crystals were crushed and seed sieves and RNA substrates were prepared by in vitro transcription. Subsequently, 2´-O-MTase assays and echo mass spectrometry analyzes were performed. The rate of MTase activity, 2′-O-MTase inhibition by sinefungin and substrate conversion rates (SAM) were determined, and the half-maximal inhibitory concentration (IC50) values ​​were determined.

The crystallographic data set of the obtained diffraction crystals was analyzed. The structural features of the VP39/sinefungin complex were examined using the molecular substitution method with the vaccinia virus VP39/SAH complex structure as a search model. To examine the enzymatic activity of recombinant VP39, two substrates with different penultimate bases (m7GpppA RNA and m7GpppG RNA) were tested.

The VP39-sinefungin interactions were analyzed by building a model of the sinefungin:RNA:VP39 complex to illustrate the molecular mechanisms underlying VP39 inhibition by sinefungin. In addition, the catalytic sites of VP39 were compared with those of 2′-O-ribose MTases from distant Zika viruses and SARS-CoV-2.

Results

The MPX structure comprised a Rossman fold similar to the alpha/beta fold (α/β), with the centrally located β-sheet spanning β2-β10 in a pattern resembling the J letter. Notably, the pattern was also found for the 2′-O MTase nonstructural protein (nsp)1614 of SARS-CoV-2. The central β-sheet was attached at one end by alpha-1, alpha2, alpha-6, and alpha-7 helices and at the other end by the alpha-3 and alpha-7 helices, and the sides were connected by β1. β11 and α5.

Both RNAS substrates were found to be acceptable; however, the one with a penultimate guanine base was preferable. Sinefungin inhibited VP39 with an IC50 value of 41 µM. Sinefungin was found to occupy the SAM pocket with its adenine base moiety located in a deep canyon lined by hydrogen-bonded hydrophobic side chains of the Val116, Phe115, Leu159, and Val139 residues. Sinefungin efficiently protected the 2′-O-ribose region with its amino groups near the 2′-ribose region where the sulfur atom of SAM would otherwise be located.

The SAM gorge had two ends, of which one end, adjacent to the RNA pocket, was crucial for positioning SAM for methyltransferase reactions, and the opposite end, located next to the adenine base of sinefungin, was unoccupied. Closer inspection revealed a complex network of water molecules at the site, connected by hydrogen bonds and bound to residues Glu118, Asn156 and Val116 as well as the adenine moiety.

Sinefungin scaffold molecules with moieties that could displace the water molecules and interact directly with the Glu118, Asn156, and Val116 residues could be exceptionally good binders because the displacement of the water molecules could produce favorable entropic effects. The similarity of the MPXV SAM binding site to Zika and SARS-CoV-2 was striking. Identical conformations were observed between sinefungin and the NS5, nsp16, and VP39 proteins of Zika, SARS-CoV-2, and MPXV, respectively.

The catalytic residue tetrad (Asp138, Lys41, Glu218, and Lys175) for MPXV was conserved among the three remote viruses tested, including the residue conformations. In addition, all viruses used an aspartate residue to interact with the amino group of sinefungin. The conserved binding modes between the three viruses suggested that a single MTase inhibitor could potentially be used as a pan-antiviral agent. However, differences in the binding modes of the nucleobases and the ribose ring were observed.

Overall, the study results showed that MTase-based inhibitors could be pan-antiviral targets.

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources

References:

• Vorläufiger wissenschaftlicher Bericht.
  Jan Silhan, Martin Klima, Dominika Chalupska, Jan Kozic, Evzen Boura. (2022). Die Struktur der 2′-O-Ribose-Methyltransferase VP39 des Affenpockenvirus im Komplex mit Sinefungin bildet die Grundlage für das Inhibitordesign. bioRxiv. doi: https://doi.org/10.1101/2022.09.27.509668 https://www.biorxiv.org/content/10.1101/2022.09.27.509668v1
• Von Experten begutachteter und veröffentlichter wissenschaftlicher Bericht.
  Silhan, Jan, Martin Klima, Tomas Otava, Petr Skvara, Dominika Chalupska, Karel Chalupsky, Jan Kozic, Radim Nencka und Evzen Boura. 2023. „Entdeckung und strukturelle Charakterisierung von Monkeypox-Virus-Methyltransferase-VP39-Inhibitoren zeigen Ähnlichkeiten mit SARS-CoV-2-Nsp14-Methyltransferase.“ Naturkommunikation 14 (1). https://doi.org/10.1038/s41467-023-38019-1. https://www.nature.com/articles/s41467-023-38019-1.

Article revisions

• 15. Mai 2023 – Das vorab gedruckte vorläufige Forschungspapier, auf dem dieser Artikel basiert, wurde zur Veröffentlichung in einer von Experten begutachteten wissenschaftlichen Zeitschrift angenommen. Dieser Artikel wurde entsprechend bearbeitet und enthält nun einen Link zum endgültigen, von Experten begutachteten Artikel, der jetzt im Abschnitt „Quellen“ angezeigt wird.