title: Molecular basis for specific viral RNA recognition and 2'-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5).
abstract: Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5' exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5' RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5' RNA cap: G0pppAG-RNA → (m7)G0pppAG-RNA ("cap-0")→(m7)G0pppAm2'-O-G-RNA ("cap-1"). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5'-(m7)G0pppA1G2U3U4G5U6U7-3'), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2'-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.
authors.affiliation: Program in Emerging Infectious Diseases, Duke-NUS (National University of Singapore) Graduate Medical School, Singapore 169857; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456;
authors.forename: Yongqian
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551; Novartis Institute for Tropical Diseases, Singapore 138670;
authors.forename: Tingjin Sherryl
authors.initials: TS
authors.lastname: Soh
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore 138670;
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551; Novartis Institute for Tropical Diseases, Singapore 138670;
authors.forename: Ka Yan
authors.initials: KY
authors.lastname: Chung
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456;
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: Program in Emerging Infectious Diseases, Duke-NUS (National University of Singapore) Graduate Medical School, Singapore 169857; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456;
authors.forename: Subhash G
authors.initials: SG
authors.lastname: Vasudevan
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore 138670; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-1055; Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1055; Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-1055; peshi@utmb.edu julien@ntu.edu.sg luodahai@ntu.edu.sg.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551; Centre d'Immunologie et des Maladies Infectieuses, Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de Médecine Pierre et Marie Curie, 75013 Paris, France; Institute of Structural Biology, Nanyang Technological University (NTU), Singapore 636921; peshi@utmb.edu julien@ntu.edu.sg luodahai@ntu.edu.sg.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: Institute of Structural Biology, Nanyang Technological University (NTU), Singapore 636921; Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 636921 peshi@utmb.edu julien@ntu.edu.sg luodahai@ntu.edu.sg.
authors.forename: Dahai
authors.identifier: http://orcid.org/0000-0001-7637-7275
authors.initials: D
authors.lastname: Luo
mesh_terms: D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012315:RNA Caps / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C582223:NS5 protein, dengue virus; D012315:RNA Caps; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases
keywords: 2′-O-ribose methyltransferase; cap-0 RNA; dengue virus; innate immunity evasion; nonstructural protein 5 methyltransferase-polymerase