Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase

Abstract

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.

Keywords: S-adenosyl methionine; flavivirus; methyltransferase; refolding.

Figure 1

Purification and CD spectrum analysis of refolded DNV3 MTase. A: SDS-PAGE gel of the purification of refolded (Rf) and native (Nt) DNV3 MTase. B: Far UV spectrum CD of native (black line), refolded (red line), and denatured (green line) DNV3 MTase. C: Temperature dependence of molar ellipticity at 222 nm for native (black) and refolded (red) DNV3 MTase. D: Near UV Spectrum CD of native (black line) and refolded (red line) DNV3 MTase.

Figure 2

ITC Analysis of SAM or SIN binding to DNV3 MTase. Heat flow versus time (upper panels) and reaction enthalpy (lower panels) at 25°C of (A) SIN injected into ITC buffer containing native DNV3 MTase, (B) SIN injected into ITC buffer containing refolded DNV3 MTase, and (C) SAM injected into ITC buffer containing refolded DNV3 MTase. The negative peaks in the upper panels represent exothermic heat of binding at each injection of SIN or SAM.

Figure 3

Endogenous SAM activity is present in native Flavivirus MTases. A: Methylation reactions were performed on WNV ³²P-labeled G*pppA-capped RNA (0.25 µM) with the indicated amount of either native (Nt) or refolded (Rf) DNV3 MTase in the presence or absence of exogenously added SAM. The methylated RNA cap (m7) migrates higher on the TLC plate than the nonmethylated cap (G*pppA). B: Quantification of the methylation reaction in (A). C: Methylation reactions performed as in (A) using 2 µg of native MTases (3 µM) from either WNV, DNV2, DNV3, or YFV in the presence or absence of exogenously added SAM. Untreated G*pppA (G) is included for comparison. D: Quantification of the methylation reaction in (C). The percentage of conversion of G*pppA to m7G*pppA was normalized in such a way that the conversion rate for each native flavivirus MTase with exogenous SAM addition was set to 100%, and the conversion rate without exogenous SAM was normalized to the one with SAM.

Figure 4

Mass Spectrometry confirms that SAM copurifies with native DNV3 MTase. A: LC-MS/MS of ETOH extraction from native WNV MTase. B: LC-MS/MS of commercial SAM standard.

Figure 5

Refolded and native DNV3 MTase have similar crystal structures. A: Alignment of the A-chains of native (magenta, PDB 3P8Z) and refolded (cyan) DNV3 MTase crystal structures. Loops where the structures differ (residues 80–84, 104–111, and 243–247) are indicated by a blue circle, red box, and green box, respectively. B: The 2Fo-Fc electron density map of the refolded DNV3 MTase SAM-binding site at 2.1 Å resolution, contoured at 1.08 σ level (0.2701 e/ų) above the mean density. SAH from the native structure B-chain is added to the SAM-binding site for reference and is colored by element, carbon, green; nitrogen, blue; oxygen, red; sulfur, yellow. C: Active sites from the aligned native (magenta) and refolded (cyan) DNV3 MTase A-chains. The SAH-derived compound present in the 3P8Z A-chain is colored as SAH in (B). D: As in (C), but SAM-binding sites are from the aligned B-chains. SAH is present in the 3P8Z B-chain and is colored by element as in (B).