Structure and Function of Flavivirus NS5 Methyltransferase<sup><a href="#fn2" rid="fn2" class=" fn">▿</a></sup>

Yangsheng Zhou

Debashish Ray

Yiwei Zhao

Hongping Dong

Wadsworth Center, New York State Department of Health,^{Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201}²

^{Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208. Phone for Pei-Yong Shi: (518) 473-7487. Fax: (518) 473-1326. E-mail:}gro.htrowsdaw@pihs. Phone for Hongmin Li: (518) 486-9154. Fax: (518) 408-2190. E-mail: gro.htrowsdaw@hil

^{These authors made equal contributions.}

Received 2006 Dec 7; Accepted 2007 Jan 22.

Abstract

The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m^{GpppAmG). 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 → m}^{GpppA → m}^{GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m}^{GpppA-RNA can be readily methylated to m}^{GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å 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.

Abstract

Eukaryotic mRNAs possess a 5′ cap structure that is cotranscriptionally formed in the nucleus. mRNA capping is essential for mRNA stability and efficient translation (13, 39). Most animal viruses that replicate in cytoplasm encode their own capping machinery to produce capped RNAs. RNA capping generally consists of three steps in which the 5′ triphosphate end of nascent RNA transcript is first hydrolyzed to a 5′ diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (15). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2′-OH position by a nucleoside 2′-O MTase, to form cap 1 (m^{GpppNm) and cap 2 (m}^{GpppNmNm) structures, respectively (}13). Both N-7 and 2′-O MTases use S-adenosyl-l-methionine (SAM) as a methyl donor and generate S-adenosyl-l-homocysteine (SAH) as a by-product. The order of capping and methylation varies among cellular and viral RNAs (13).

The genus Flavivirus comprises approximately 70 viruses, many of which are important human pathogens, including four serotypes of dengue virus (DENV), yellow fever virus (YFV), St. Louis encephalitis virus, and West Nile virus (WNV) (23). The flavivirus genome is a single-stranded RNA of positive (i.e., mRNA sense) polarity. The 5′ end of the genome contains a type 1 cap followed by a conserved dinucleotide sequence 5′-AG-3′ (7, 41). The single open reading frame of the flavivirus genome encodes a polyprotein, which is processed by viral and cellular proteases into three structural proteins and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (23). Of the four enzymes required for synthesis of flavivirus m^{GpppAm cap structure, the RNA triphosphatase and 2′-O MTase have been, respectively, mapped to NS3 (}20, 42) and NS5 (9). We recently showed that WNV NS5 carries both guanine N-7 and ribose 2′-O MTase activities (34). The guanylyltransferase for flavivirus capping remains elusive.

Flavivirus NS5 consists of an N-terminal MTase and a C-terminal RNA-dependent-RNA polymerase (RdRp) domain (1, 16, 28). The structure of DENV-2 MTase suggests that flavivirus NS5 MTase belongs to a family of SAM-dependent MTases (9). Most of the MTases within this family, including both N-7 and 2′-O RNA MTases such as Encephalitozoon cuniculi (Ecm1) N-7 MTase and vaccinia virus 2′-O MTase VP39 (10, 18), share a common core structure referred to as a “SAM-dependent MTase fold,” composed of an open α/β/α sandwich structure (11, 24). Structure and sequence comparisons of the 2′-O MTases suggest that a conserved K-D-K-E tetrad forms the active site for the 2′-O methyl transfer reaction (9). Using Ala substitution, we recently showed that all residues within the K₆₁-D₁₄₆-K₁₈₂-E₂₁₈ tetrad of the WNV MTase are essential for 2′-O methylation activity, whereas D₁₄₆ is more critical than the other three residues for N-7 methylation. In addition, we found that methylations of guanine N-7 and ribose 2′-O of the WNV cap structure are sequential, with N-7 preceding 2′-O methylation (34). The WNV MTase represents a unique system to study how a single enzyme catalyzes two distinct cap methylations.

Here we report that, similar to the WNV, MTases from other flaviviruses also sequentially methylate viral RNA cap at guanine N-7 and ribose 2′-O positions, indicating that it is a general mechanism for flaviviruses to encode the NS5 MTase with dual methylation activities for an efficient synthesis of the viral RNA cap. By contrast, the crystal structure of the WNV MTase in complex with SAH shows only a single SAM-binding site. Thus, the 5′ cap of flavivirus RNA must evidently be repositioned to accept two methyl groups from SAM during methylations. Biochemical and mutagenesis analyses suggest that the WNV MTase methylates the N-7 and 2′-O positions using two distinct mechanisms. In the context of full-length WNV, a mutation (D₁₄₆A) defective in both the N-7 and 2′-O methylations is lethal to the virus. Mutant viruses inactive for 2′-O but not N-7 methylation (K₆₁A, K₁₈₂A, or E₂₁₈A) are attenuated in cell culture and in mice and can be used to protect mice from challenge with wild-type WNV.

Acknowledgments

We are grateful to Kiong Ho for providing recombinant VP39 protein and for helpful discussions and to Corey Bennett and Aaloki Shah for technical assistance. We thank the Molecular Genetics Core, the Cell Culture Core, Mass Spectrometry and Proteomics Core, and the Macromolecular Crystallography Facility at the Wadsworth Center for DNA sequencing for maintenance of BHK and Vero cells, for verification of mutant MTases, and for crystal screening, respectively.

The work was partially supported by contract AI25490 and grants AI061193 and AI065562 from NIH. The BSL-3 animal facility at the Wadsworth Center was used, which is funded in part by the Northeast Biodefense Center's animal core (NIH/NIAID U54 AI05 7158). D.R. is supported by a postdoctoral fellowship from the National Sciences and Engineering Research Council of Canada. X-ray diffraction data for this study were measured at beamline X4A of the national synchrotron light source, which is supported by the Department of Energy, by grants from the NIH, and by the New York Structural Biology Center.

Acknowledgments

Footnotes

^{Published ahead of print on 31 January 2007.}

Footnotes

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