Structure and function of flavivirus NS5 methyltransferase

Abstract

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

FIG. 1.

Crystal structure of the WNV MTase and comparison with the DENV-2 MTase. (A) Ribbon representation of the crystal structure of the WNV MTase. The MTase structure is colored as follows: N-terminal domain, red; MTase core, green; C-terminal domain, cyan. The bound SAH is shown in ball-and-stick representation with atom colors as follows: carbon, yellow; oxygen, red; nitrogen, blue; sulfur, green. (B) A representative omit (F_o-F_c) electron density map (magenta) showing the bound SAH and its interactions with the MTase residues. Hydrogen bonds are shown as orange dashed lines. (C) Superposition of the crystal structures of the DENV-2 (2) (pink) and WNV (cyan and red) MTases. Ribavirin (occupying the putative GTP cap-binding site for the 2′-O methylation) (see Fig. 8) and SAH are shown in ball-and-stick representation. The loops of the WNV structure that show significant differences relative to the DENV-2 structure are colored in red. (D and E) Solvent-accessible molecular GRASP (27) surface representation of the electrostatic potential of the WNV (D) and DENV-2 (E) MTases, showing the putative RNA substrate binding site. The surface is colored blue for positive (15 kT), red for negative (−15 kT) and white for neutral, where k is the Boltzmann constant and T is the temperature (27). In panels D and E, ribavirin and SAH are in stick representation with atom colors as follows: oxygen, red; nitrogen, blue; carbon, white; sulfur, green.

FIG. 2.

Flavivirus NS5 sequentially methylates guanine N-7 and ribose 2′-OH of the viral RNA cap. (A) Sequence alignment of flavivirus MTases. The MTase sequences of WNV, DENV-1, DENV-2, and YFV are derived from GenBank accession numbers AF404756, DVU88535 U87411 and YFU17-66, respectively. The alignment was performed using GCG software (Genetics Computer Group). Identical amino acids among all MTases are shaded. The conserved K₆₁-D₁₄₆-K₁₈₂-E₂₁₈ residues mutated in this study are indicated by an asterisk. The exact sequences of the recombinant DENV-1 and YFV MTases (not including the C-terminal His tag) are shown. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of recombinant MTase proteins of YFV, DENV-1, and WNV. (C) Guanine N-7 methylation activity. Substrate G*pppA-RNA was methylated with the indicated MTases or no enzyme (Mock) in the presence of cold SAM in the N-7 assay buffer for 30 min. The reaction mixtures were then digested by TAP and analyzed on a TLC plate followed by autoradiography. (D) Ribose 2′-O methylation activity. m⁷G*pppA-RNA was methylated by the indicated MTases and digested by nuclease P1. The nuclease P1-resistant cap structures were then analyzed on a TLC plate. (E) Time course analyses of the DENV-1 and YFV MTase activities. ³²P-labeled G*pppA-RNA was methylated by DENV-1 (left panel) and YFV MTase (right panel) in the 2′-O methylation buffer for the indicated times, digested with nuclease P1, and analyzed by TLC and autoradiography. The 2′-O methylation buffer is optimal for 2′-O methylation and also supports N-7 methylation. The positions of the origin and the migrations of G*p, m⁷G*p, G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are indicated on the left.

FIG. 3.

Optimization of conditions for the WNV N-7 and 2′-O cap methylations. Reactions for N-7 (•) and 2′-O methylation (○) contained substrate G*pppA-RNA and m⁷G*pppA-RNA and were incubated for 5 and 60 min, respectively. The optimal conditions were determined by individually titrating pH (A), temperature (B), MgCl₂ (C), and NaCl (D), while keeping the other three parameters constant at the optimal levels. The reaction mixtures were then treated with nuclease P1, analyzed on TLC plates, and quantified by PhosphorImager analysis. For each parameter, relative activities were presented using the optimal level as 100%. Average results from two independent experiments are shown.

FIG. 4.

Methylation activities of the WNV K-D-K-E mutant MTases. (A) Superposition of the active site residues K-D-K-E of the VP39 and WNV MTases. SAH and K-D-K-E residues of the WNV MTase are in ball-and-stick representation with carbon colored yellow. The carbon atoms of Gppp-RNA and K-D-K-E residues from VP39 are colored in pink. Colors for other atoms are as follows: phosphate, cyan; sulfur, green; oxygen, red; nitrogen, blue. (B and C) Mutant MTases (1 μg) containing the indicated substitutions were assayed for 2′-O (B) and N-7 (C) methylation activities. The experimental details are described in Materials and Methods. ³²P-labeled markers, m⁷G*pppA and G*pppA, are indicated on top (B). The relative conversions for 2′-O methylation (m⁷G*pppA to m⁷G*pppAm in panel B) and for N-7 methylation (G*pppA to m⁷G*pppA in panel C) were calculated by comparing the products generated from the mutant MTases with that produced from the wild-type protein (set at 100%). For N-7 methylation (C), the reaction mixtures were incubated for 5 min (top panel) or 30 min (TLC data not shown); the relative activities between the mutants and wild type for both incubation times are summarized (bottom panel). Average results from two to three experiments are shown.

FIG. 5.

Comparison of specific infectivity, RNA replication, protein synthesis, and virus production in cells transfected with genome-length RNA containing K-D-K-E mutations. Equal amounts of wild-type and mutant genome-length RNAs were transfected into BHK cells and assayed for specific infectivity (A). At indicated time points, viral RNA replication, protein expression, and virus production were monitored by real-time RT-PCR (B), IFA (C), and plaque assay (D), respectively. For specific infectivity, an average result of four independent experiments is shown (A). One representative result of two independent experiments is shown for real-time RT-PCR (B), IFA (C), and viral yield production (D). Viral RNA synthesis (B) is presented as RNA copy number per 100 ng of total cellular RNA, using full-length WNV RNA transcribed from an infectious cDNA clone (38) as a reference. Virus production (D) in the supernatants of transfected cells was quantified by plaque assays on Vero cells.

FIG. 6.

Plaque morphology and growth kinetics of the wild-type and K-D-K-E mutant viruses. (A) Plaque morphologies of wild-type and mutant viruses on Vero cells. A single- or double-nucleotide change was engineered into the genome-length RNA to introduce an Ala substitution within the K-D-K-E motif. Wild-type and K₆₁A (AAA to GCA; mutated nucleotide underlined), K₁₈₂A (AAG to GCG), and E₂₁₈A (GAG to GCG) mutant viruses were harvested on day 5 p.t. “Mutant” D₁₄₆A (GAC to GCC) virus was recovered starting on day 8 p.t. Plaques were developed at 96 h p.i. Sequences of the mutated regions, derived from the harvested viruses, are presented below the plaque assay plates. (B and C) Growth kinetics of the wild-type, K₆₁A, K₁₈₂A, and E₂₁₈A viruses were compared in Vero cells (B) and C6/36 cells (C) by infecting cells with an MOI of 0.1, followed by quantification of viral titers using plaque assay on Vero cells.

FIG. 7.

A D₁₄₆E mutation of the K-D-K-E motif could support the WNV replication and the N-7 MTase activity. (A) A genome-length RNA containing a double-nucleotide mutation D₁₄₆A₂ (GAC to GCA) was transfected into BHK cells. Viruses in culture fluids were harvested on day 5 and 9 p.t. and assayed for plaque morphologies on Vero cells. The large plaques derived from day 9 p.t. were amplified and are shown. For each virus, the sequences of the mutated NS5 regions were presented below the plaque morphology. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the D₁₄₆E mutant. (C and D) The D₁₄₆E MTase was assayed for the N-7 (C) and 2′-O methylation activities (D) using substrate G*pppA-RNA and m⁷G*pppA-RNA, respectively. The relative activities between the wild-type (set as 100%) and mutant D₁₄₆E are presented below the TLC images in panels C and D. The N-7 reactions in panel C were incubated in N-7 methylation buffer for 5 min. The positions of the origin and the G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are indicated on the left of the TLC plates.

FIG. 8.

Model for the sequential guanine N-7 and ribose 2′-OH methylations of the RNA cap by flavivirus MTase. The SAM- and m⁷G-binding sites are shaded and depicted by an oval and a rectangle, respectively. The 5′-terminal nucleotides of the WNV genome, GpppAGUA-RNA, are shown.