In a recent study published on the bioRxiv* preprint server, researchers explored the crystal structure of monkeypox virus (MPX) (MPXV) and the complex of VP39, a 2′-O-RNA methyltransferase (MTase) and sinefungin, a pan- MTase inhibitor.
Study: Structure of monkeypox virus 2′-O-ribose methyltransferase VP39 in complex with sinefungin provides basis for inhibitor design. Image credit: Marina Demidiuk/Shutterstock
The number of cases of MPX is increasing every hour worldwide and could indicate a new pandemic. The structural analysis of MPXV could help in the development of effective antiviral agents to combat MPXV. Poxviruses encode decapping-type enzymes to prevent the accumulation of double-stranded ribonucleic acid (dsRNA) during infection that could induce innate antiviral immune responses. MPXV encodes the toxin enzyme that inhibits the cGAS-STING (cyclic GMP-AMP synthase stimulator of interferon genes) pathway activated by ds-deoxyribonucleic acid (dsDNA).
Methylation of the initial nucleotide (nt) of the mature MPXV cap (or cap-1) at the 2′-O ribose location has been documented. MTase is required by the poxviridiae virus family (including MPXV) for cap-0 synthesis, and by adding another methyl group to the 2′-O location of the proximal ribose, the immature cap (cap-0) can become the mature none The step is essential to prevent the development of innate immune responses and is catalyzed by VP39, the 2′-O MTase of MPXV.
About the study
In the present study, the researchers evaluated the structure of the VP39-sinefungin complex of MPXV to improve the understanding of the mechanisms of inhibition of the VP39 molecule by sinefungin. They also compared the structure to the 2′-O MTases of single-stranded RNA (ssRNA) viruses such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The VP39 gene of MPXV strain USA-May22 was codon-optimized for expression in E. coli for subsequent synthesis and cloning. BL21 cells of E. coli were transformed with a plasmid expressing VP39 and IPTG (isopropyl-bD-thiogalac-topyranoside) was added, after which the recombinant VP39 was purified. The cells were centrifuged, lysed, and the lysate subjected to chromatography analysis. VP39 was concentrated and mixed with sinefungin for crystallization-based assays.
Initially formed crystals were crushed and seeding screens and RNA substrates were prepared by in vitro transcription. Subsequently, 2´-O-MTase assays and echo mass spectrometry analyzes were performed. MTase activity rate, 2′-O-MTase inhibition by sinefungin and substrate conversion rates (SAM) were determined and half maximal inhibitory concentration (IC50) values were determined. .
The crystallographic data set of the diffraction crystals obtained was analyzed. The structural features of the VP39/sinefungin complex were studied using the molecular replacement method with the structure of the vaccinia virus VP39/SAH complex as a search model. To verify the enzymatic activity of recombinant VP39, two substrates with different penultimate bases (m7GpppA-RNA and m7GpppG-RNA) were tested.
VP39-sinefungin interactions were analyzed by constructing a model of the sinefungin:RNA:VP39 complex to illustrate the molecular mechanisms underlying VP39 inhibition by sinefungin. In addition, VP39 catalytic sites were compared with those of 2′-O-ribose MTases from distant Zika viruses and SARS-CoV-2.
results
The MPX structure included a Rossman fold similar to the alpha/beta (α/β) fold, with the centrally located β-sheet consisting of β2-β10 in a pattern resembling the letter J. In particular, it also pattern was found for the non-structural protein 2′-O MTase (nsp) 1614 of SARS-CoV-2. The central β-sheet was held in place at one end by helices alpha-1, alpha2, alpha-6, and alpha-7 and by helices alpha-3 and alpha-7 at the other end, and the sides were connected by β1, β11 and α5.
Both RNAS substrates were found to be acceptable; however, the 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 barrel located flanked by hydrophobic-type side chains of hydrogen-bonded residues Val116, Phe115, Leu159, and Val139. 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 barrel had two ends, of which one end bordering the RNA pocket was vital for positioning SAM for methyltransferase reactions, and the opposite end adjacent to the sinefungin adenine base was unoccupied. On closer inspection, the location showed a complex network of water molecules connected by hydrogen bonds and attached to residues Glu118, Asn156 and Val116 and the adenine moiety.
Molecules based on Sinefungin scaffolds containing moieties that could displace water molecules and directly interact with residues Glu118, Asn156, and Val116 could be exceptionally good binders, as the displacement of water molecules could cause favorable entropic effects . The similarity of the MPXV SAM binding site to Zika and SARS-CoV-2 was remarkable. Identical conformations were observed between sinefungin and NS5, nsp16, and VP39 proteins from Zika, SARS-CoV-2, and MPXV, respectively.
The tetrad of catalytic residues (Asp138, Lys41, Glu218, and Lys175) for MPXV was conserved among the three distant viruses tested, including residue conformations. In addition, all viruses used an aspartate residue to interact with the amino group of sinefungin. The conserved binding modes among the three viruses indicated that a single MTase inhibitor could potentially be used as a pan-antiviral agent. However, differences in the binding modes of the nucleobase and ribose ring were observed.
Overall, the findings of the study demonstrated that MTase-based inhibitors could be pan-antiviral targets.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.