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 muse for inhibitor design. Image Credit: Marina Demidiuk/Shutterstock

MPX case counts are rising by the hour across the globe and will indicate a latest pandemic. Structural evaluation of MPXV could aid in the event of effective antiviral agents to combat MPXV. Poxviruses encode decapping-type enzymes for stopping double-stranded ribonucleic acid (dsRNA) accumulation during infection that might 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 two′-O ribose location has been documented. MTase is required by the poxviridiae family of viruses (including MPXV) for cap-0 synthesis and by adding one other methyl group at the two′-O location of the proximal ribose, the immature cap (cap-0) will be converted to the mature cap. The step is important for stopping the event of innate immune responses and is catalyzed by VP39, the two′-O MTase of MPXV.

In regards to the study

In the current study, researchers assessed the VP39-sinefungin complex structure of MPXV to enhance understanding of the mechanisms of VP39 molecule inhibition by sinefungin. In addition they compared the structure to 2′-O MTases of single-stranded RNA (ssRNA) viruses resembling 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 evaluation. 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 speed 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 in comparison with that of  2′-O-ribose MTases from distant Zika viruses and SARS-CoV-2.

Results

The MPX structure included a Rossman fold resembling alpha/beta (α/β) folding, with the centrally situated β-sheet comprising β2-β10 in a pattern resembling the J letter. Notably, the pattern was also found for the two′-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 opposite end, and the edges were connected by β1, β11 and α5.

Each the RNAS substrates were found to be acceptable; nonetheless, 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 situated canyon lined by hydrophobic-type sidechains of the Val116, Phe115, Leu159, and Val139 residues with hydrogen bonding. Sinefungin efficiently protected the two′-O-ribose region with its amino groups near the two′ ribose region where SAM’s sulphur atom could 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 other end situated beside the sinefungin’s adenine base was unoccupied. On closer inspection, the placement showed a posh water molecule network connected by hydrogen bonding and certain to the Glu118, Asn156, and Val116 residues and the adenine moiety.  

Sinefungin scaffold-based molecules bearing moieties that might displace the water molecules and directly interact with the Glu118, Asn156, and Val116 residues could possibly 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. Similar conformations were observed amongst 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 many three distant viruses tested, including the residue conformations. Further, all of the viruses used an aspartate residue for interacting with sinefungin’s amino group. The conserved binding modes among the many three viruses indicated that a single MTase inhibitor could possibly be potentially used as a pan-antiviral agent. Nevertheless, differences were observed within the nucleobase and ribose ring binding modes.

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

*Vital notice

bioRxiv publishes preliminary scientific reports that usually are not peer-reviewed and, due to this fact, mustn’t be thought to be conclusive, guide clinical practice/health-related behaviour, or treated as established information.