Abstract
The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2'-O positions of the viral RNA cap (GpppA-RNA → m(7)GpppA-RNA → m(7)GpppAm-RNA), using S-adenosyl-l-methionine (AdoMet) as a methyl donor. We report here that sinefungin (SIN), an AdoMet analog, inhibits several flaviviruses through suppression of viral MTase. The crystal structure of West Nile virus MTase in complex with SIN inhibitor at 2.0-Å resolution revealed a flavivirus-conserved hydrophobic pocket located next to the AdoMet-binding site. The pocket is functionally critical in the viral replication and cap methylations. In addition, the N7 methylation efficiency was found to correlate with the viral replication ability. Thus, SIN analogs with modifications that interact with the hydrophobic pocket are potential specific inhibitors of flavivirus MTase.
Highlights
The genus Flavivirus in the family Flaviviridae is composed of more than 70 viruses (1)
Effects of the Additional Pocket on West Nile virus (WNV) Replication—To val- viously found that the N7 methylation activity is essential for idate the biological relevance of the identified pocket, we per- the WNV life cycle, the viral methyltransferase represents a formed mutagenesis using a luciferase-reporting replicon of novel target for flavivirus therapy (19, 20, 28, 29, 47)
The replicon contained a luciferase reporter in- SIN was previously shown to inhibit WNV in cell culture frame fused with the viral open reading frame (ORF), in the position where structural with EC50 and CC50 values of 23 M and 4.5 mM, respectively genes were deleted (Fig. 4A) (38)
Summary
Expression, and Purification of the WNV, WNV Mutant, YFV, and DENV-2 MTase—The WNV MTase domain containing the N-terminal 300 amino acids of NS5 was prepared for co-crystallization and enzyme assays as described previously (19, 20). Crystallization, X-ray Data Collection, Structure Determination, and Refinement—Co-crystals of the MTase1⁄7SIN complex were grown by methods described previously (20), except in the presence of 5 mM SIN. In Vitro MTase Inhibition Assay—The N7 and 2Ј-O-methylation inhibition assays were performed as described previously for WNV (28, 41), except that the 5Ј-end-labeled substrates G*pppA-RNA and m7G*pppA-RNA, representing the first 211 nucleotides of the DENV-2 genome (the asterisk indicates that the following phosphate was 32P-labeled), were used. The 2Ј-O-methylation was monitored by conversion of m7G*pppA-RNA 3 m7G*pppAm-RNA Both methylation assays were performed with 0.25 g of DENV-2 MTase, 25 M AdoMet, and various concentrations of SIN. Showed no cytotoxicity when uninfected cells were treated with SIN at concentrations up to 300 M, indicating that the observed antiviral activity was not due to compoundmediated cytotoxicity (Fig. 1C)
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