Identification and Characterization of a Ribose 2′-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales

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   FIG 1

   Identification of nidovirus 2′-O-MTases. (A) Domain organization of the replicase pp1ab polyprotein for selected nidoviruses: SARS-CoV (coronavirus), EToV (torovirus), and GAV (ronivirus). The predicted domains are indicated, and the predicted cleavage sites are marked with arrowheads. The domains are as follows: ADP-ribose-1′-phosphatase (ADRP), papain-like proteinases (PLpro), chymotrypsin-like proteinase (3CLpro), RNA-dependent RNA polymerase (RdRp), helicase (Hel), exonuclease (ExoN), N7-methyltransferase (N7MT), uridylate-specific endoribonuclease (Ne; also abbreviated NendoU), and 2′-O-methyltransferase (2OMT). The coronavirus nsp10 and its similarly located counterparts in EToV and GAV are depicted in purple, and RFS stands for the ribosomal frameshift site. The start sites and endpoints of the expressed proteins are indicated. (B) Expression and purification of recombinant viral proteins. The gels of SDS-PAGE were stained with Coomassie brilliant blue. GAV nsp10, GAV nsp16, and EToV nsp16 are GST fusion proteins, and the others were 6-histidine tagged. These protein bands are indicated with black arrows. (C) Activities of potential 2′-O-methyltransferases in ³H-methyl incorporation assays. Vaccinia virus VP39 and SARS-CoV nsp10/nsp16 were used as positive controls, while SARS-CoV nsp10 acted as a negative control for 2′-O-MTase activity. The final concentration of the nsp16 proteins of SARS-CoV, GAV, and EToV was 1 μM. The cpm values reflect the 2′-O-MTase activity that was detected by liquid scintillation counting. The error bars indicate standard deviations.
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   FIG 2

   RNA substrate specificity of GAV nsp16 2′-O-MTase. (A) (Lane 1) An RNA representing the first 68 nucleotides of the GAV genome was capped to form ^7MeG*pppA-RNA, incubated with GAV nsp16, digested by nuclease P1 to release cap structures, and analyzed by TLC. (Lanes 2 to 4) Cap analogs used as controls were generated by vaccinia virus capping enzymes D1/D12 in the absence of SAM (lane 2), in the presence of SAM (lane 3), and with SAM and VP39 2′-O-MTase (lane 4). (B) SAM-dependent methyltransferase activities of GAV nsp16 and SARS nsp16. Equal amounts of proteins were incubated with AC₂₀ (white), GpppAC₂₀ (gray), and ^7MeGpppAC₂₀ (black) in the presence of ³H-labeled SAM for 1 h, and radioactivity incorporation was detected by liquid scintillation counting. (C) MTase activities of GAV nsp16 for capped RNAs with different initiating nucleotides. ^7MeGpppAC₂₀, ^7MeGpppCC₂₀, ^7MeGpppUC₂₀, ^7MeGpppGC₂₀, and ^7MeGpppA were used as substrates to test GAV nsp16 activity. (D) GAV nsp16 2′-O-MTase activities on capped RNA substrates of different lengths. The activity was formulated as a percentage (100% corresponds to the activity of ^7MeGpppAC₂₀). The error bars indicate standard deviations.
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   FIG 3

   Determining the optimal reaction conditions for GAV nsp16. The GAV nsp16 activity was measured in the presence of ³H-labeled SAM by liquid scintillation counting of transferred ³H-methyl. (A) Effect of temperature on enzymatic activity. Reactions were performed in Tris buffer (pH 8.0) with incubation at various temperatures. RNAs were purified with Sephadex A-50 and detected by liquid scintillation counting. (B) Enzymatic activities of GAV nsp16 at different pH values, including citric acid-NaOH (pH 6.0), Tris-HCl buffer (pH 7.0 to 9.0), and Na₂CO₃-NaOH buffer (pH 10 and 11). An activity of 100% corresponds to that at pH 8.0. (C) Influence of positive-valence metal ions on enzymatic activity. The reactions were conducted at pH 8.0 and 20°C. The error bars indicate standard deviations.
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   FIG 4

   Sequence alignment and homology modeling of GAV nsp16. (A) Multiple-sequence alignment of selected 2′-O-methyltransferases of nidoviruses. The β-strands of the canonical MTase fold are labeled from β1 to β7, and additional β-strands from B1 onward. (B) Ribbon representation of the GAV nsp16 model structure, generated based on the template structure of SARS nsp10/nsp16 (PDB accession no. 3R24). (C) SAM was added to the GAV nsp16 model by superimposition of the model and the structure of SARS nsp10/nsp16 (PDB accession no. 3R24). The β-strands were marked in accordance with the alignment. (D) Schematic diagrams of the topologies of GAV nsp16 (modeled structure) and SARS nsp16. (E) Surface electrostatic potentials of GAV nsp16 (model) and SARS nsp16. The surface electrostatic potential diagram was generated by PyMol; the blue areas represent positively charged areas, while the red areas represent negatively charged areas.
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   FIG 5

   Activities of wild-type (WT) GAV nsp16 and K-D-K-E mutants. (A) MTase activities of GAV nsp16 (WT and mutants) detected by using ^7MeGpppAC₂₀ as the substrate in ³H-methyl incorporation assays. (B) MTase activities of GAV nsp16 (WT and mutants) detected by using ^7MeGppp-RNA (GAV 5′ UTR) as the substrate in ³H-methyl incorporation assays. (C) MTase activities of GAV nsp16 (WT and mutants) analyzed by RNA digestion and TLC assays. Lane 4 represents the WT of GAV nsp16; lane 2 is a negative control, lane 3 is a positive control treated with vaccinia virus VP39, and lanes 5 to 12 are mutants of GAV nsp16. The error bars indicate standard deviations.
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   FIG 6

   Inhibition of GAV nsp16 2′-O-MTase activity by established methyltransferase inhibitors. Increasing concentrations of sinefungin (A), ribavirin (B), and AdoHcy (a by-product of the reaction) (C) were added to the reaction mixtures, and the activity was measured by using ³H-methyl incorporation MTase activity assays. The error bars indicate standard deviations.

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