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

Clinical Trials & Research

In a recent study posted to the bioRxiv* preprint server, researchers explored the crystalline structure of the monkeypox (MPX) virus (MPXV) and the complex of VP39, a 2′-O-RNA methyltransferase (MTase) and sinefungin, a pan-MTase inhibitor.

Study: The structure of monkeypox virus 2’-O-ribose methyltransferase VP39 in complex with sinefungin provides the foundation for inhibitor design. Image Credit: Marina Demidiuk/Shutterstock

MPX case counts are rising by the hour across the globe and could indicate a new pandemic. Structural analysis of MPXV could aid in the development of effective antiviral agents to combat MPXV. Poxviruses encode decapping-type enzymes for preventing double-stranded ribonucleic acid (dsRNA) accumulation during infection that could induce innate antiviral immune responses. MPXV encodes the poxin enzyme that inhibits the ds deoxyribonucleic acid (dsDNA)-triggered cGAS-STING (Cyclic GMP-AMP synthase- stimulator of interferon genes) pathway.

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 family of viruses (including MPXV) for cap-0 synthesis and by adding another methyl group at the 2′-O location of the proximal ribose, the immature cap (cap-0) can be converted to the mature cap. The step is essential 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 assessed the VP39-sinefungin complex structure of MPXV to improve understanding of the mechanisms of VP39 molecule inhibition by sinefungin. They also compared the structure to 2′-O MTases of single-stranded RNA (ssRNA) viruses such as the Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The MPXV USA-May22 strain VP39 gene was codon-optimized to be expressed in E. coli for subsequent synthesis and cloning. E. coli BL21 cells were converted with VP39-expressing plasmid and IPTG (isopropyl-b-D-thiogalac- topyranoside) was added, following which the recombinant VP39 was purified. The cells were centrifuged, lysed, and the lysate was subjected to chromatography analysis. VP39 was concentrated and mixed with sinefungin for crystallization-based trials.

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

The crystallographic dataset of the obtained diffraction crystals was analyzed. The VP39/sinefungin complex structural characteristics were studied using the molecular substitution method with the vaccinia virus VP39/SAH complex structure as a search model. For verifying recombinant VP39’s enzymatic activity, two substrates with differing penultimate bases (m7GpppA-RNA and m7GpppG-RNA) were tested.

The VP39-sinefungin interactions were analyzed by constructing a model of the sinefungin:RNA:VP39 complex for illustrating the molecular mechanisms underlying VP39 inhibition by sinefungin. Further, VP39 catalytic sites were compared to that of  2′-O-ribose MTases from distant Zika viruses and SARS-CoV-2.


The MPX structure included a Rossman fold resembling alpha/beta (α/β) folding, with the centrally located β-sheet comprising β2-β10 in a pattern resembling the J letter. Notably, the pattern was also found for the 2′-O MTase non-structural protein (nsp)1614 of SARS-CoV-2. The central β sheet was secured in place from one end by 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 the RNAS substrates were found to be acceptable; however, the one with a guanine penultimate 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 situated in a deeply located canyon lined by hydrophobic-type sidechains of the Val116, Phe115, Leu159, and Val139 residues with hydrogen bonding. Sinefungin efficiently protected the 2′-O-ribose region with its amino groups near the 2′ ribose region where SAM’s sulphur atom would be situated otherwise.

The SAM canyon had two ends, of which one end bordering the RNA pocket was vital for positioning SAM for methyltransferase reactions, and the opposite end located beside the sinefungin’s adenine base was unoccupied. On closer inspection, the location showed a complex water molecule network connected by hydrogen bonding and bound to the Glu118, Asn156, and Val116 residues and the adenine moiety.  

Sinefungin scaffold-based molecules bearing moieties that could displace the water molecules and directly interact with the Glu118, Asn156, and Val116 residues could be exceptionally good binders since displacing the water molecules could cause favorable entropic effects. MPXV SAM binding site resemblance with Zika and SARS-CoV-2 was remarkable. Identical conformations were observed among 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 the residue conformations. Further, all the viruses used an aspartate residue for interacting with sinefungin’s amino group. The conserved binding modes among the three viruses indicated that a single MTase inhibitor could be potentially used as a pan-antiviral agent. However, differences were observed in the nucleobase and ribose ring binding modes.

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

*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 behaviour, or treated as established information.

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