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 inhibited VP39 with an IC 50 value of 41 µM. 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.īoth the RNAS substrates were found to be acceptable however, the one with a guanine penultimate base was preferable. Notably, the pattern was also found for the 2′-O MTase non-structural protein (nsp)1614 of 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. Further, VP39 catalytic sites were compared to that of 2′-O-ribose MTases from distant Zika viruses and SARS-CoV-2.
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.
The initially formed crystals were crushed, and seeding screens and RNA substrates were prepared by transcription in vitro. VP39 was concentrated and mixed with sinefungin for crystallization-based trials. The cells were centrifuged, lysed, and the lysate was subjected to chromatography analysis. 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. coli for subsequent synthesis and cloning. The MPXV USA-May22 strain VP39 gene was codon-optimized to be expressed in E. 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). 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. The step is essential for preventing the development of innate immune responses and is catalyzed by VP39, the 2′-O MTase of MPXV. 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. Methylation of the initial nucleotide (nt) of the mature MPXV cap (or cap-1) at the 2′-O ribose location has been documented. MPXV encodes the poxin enzyme that inhibits the ds deoxyribonucleic acid (dsDNA)-triggered cGAS-STING (Cyclic GMP-AMP synthase- stimulator of interferon genes) pathway. Poxviruses encode decapping-type enzymes for preventing double-stranded ribonucleic acid (dsRNA) accumulation during infection that could induce innate antiviral immune responses. Structural analysis of MPXV could aid in the development of effective antiviral agents to combat MPXV.
MPX case counts are rising by the hour across the globe and could indicate a new pandemic. Image Credit: Marina Demidiuk/Shutterstock
Study: The structure of monkeypox virus 2’-O-ribose methyltransferase VP39 in complex with sinefungin provides the foundation for inhibitor design. 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. By Pooja Toshniwal Paharia Reviewed by Aimee Molineux
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