In a recent study published on 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 .
The number of MPX cases is increasing by the hour around the world and could indicate a new pandemic. Structural analysis of MPXV could aid in the development of effective antiviral agents to combat MPXV. Poxviruses encode knock-out-like enzymes to prevent double-stranded ribonucleic acid (dsRNA) accumulation during infection that could induce innate antiviral immune responses. MPXV encodes the enzyme poxin 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 location has been documented. MTase is required by the poxviridia 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 be converted into a mature cap. This 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, researchers evaluated the complex VP39-sinefungin 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 of single-stranded RNA (ssRNA) viruses such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The VP39 gene from strain MPXV USA-May22 was codon-optimized to be expressed in E. coli for further synthesis and cloning. E. coli BL21 cells were converted with the plasmid expressing VP39 and IPTG (isopropyl-bD-thiogalactopyranoside) was added, after which the recombinant VP39 was purified. The cells were centrifuged, lysed and the lysate was subjected to chromatographic analysis. VP39 was concentrated and mixed with sinefungin for crystallization-based assays.
The initially formed crystals were ground and seed screens and RNA substrates were prepared by transcription in vitro. Subsequently, 2´-O-MTase assays and echo mass spectrometry analyzes were performed. MTase activity level, 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 dataset of the obtained diffraction crystals was analyzed. The structural characteristics of the VP39/sinefungin complex were studied using the molecular substitution method with the structure of the vaccinia virus VP39/SAH complex as a research 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. Additionally, VP39 catalytic sites were compared to those of 2′-O-ribose MTases from distant Zika viruses and SARS-CoV-2.
The MPX structure included a Rossman fold resembling an alpha/beta (α/β) fold, with the centrally located β-sheet comprising β2-β10 in a pattern resembling the letter J. Notably, the pattern was also found to the non-structural protein 2′-O MTase (nsp) 1614 of SARS-CoV-2. The central β-sheet was fixed in place at one end by the alpha-1, alpha2, alpha-6 and alpha-7 helices and by the alpha-3 and alpha-7 helices at the other end, 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 deeply located canyon lined with hydrophobic-like side chains of residues Val116, Phe115, Leu159, and Val139 with hydrogen bonding. Sinefungin effectively 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 canyon had two ends, one end of which bordering the RNA pocket was vital for positioning SAM for methyltransferase reactions, and the opposite end located next to the adenine base of sinefungin was unoccupied. Upon closer inspection, the location showed a complex web of water molecules connected by hydrogen bonding and bound to residues Glu118, Asn156 and Val116 and the adenine moiety.
Molecules based on sinefungin scaffold bearing moieties that could displace water molecules and directly interact with residues Glu118, Asn156 and Val116 could be exceptionally good binders since displacement of water molecules could cause entropic effects favorable. The resemblance of the MPXV SAM binding site to Zika and SARS-CoV-2 was remarkable. 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 distant viruses tested, including residue conformations. Additionally, 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 were observed in the binding modes of the nucleobase and ribose ring.
Overall, the study results showed that MTase-based inhibitors could be pan-antiviral targets.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.
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