title: Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner.
abstract: Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.
authors.affiliation: Institut Pasteur of Shanghai, Chinese Academy of Sciences, 200025 Shanghai, People's Republic of China.
authors.forename: Leiyun
authors.initials: L
authors.lastname: Weng
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hirata
authors.forename: Masaaki
authors.initials: M
authors.lastname: Arai
authors.forename: Michinori
authors.initials: M
authors.lastname: Kohara
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Koichi
authors.initials: K
authors.lastname: Watashi
authors.forename: Kunitada
authors.initials: K
authors.lastname: Shimotohno
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.forename: Jin
authors.initials: J
authors.lastname: Zhong
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyoda
mesh_terms: D004789:Enzyme Activation; D005838:Genotype; D016174:Hepacivirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C / Q000378:metabolism* / Q000821:virology; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013109:Sphingomyelins / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D013109:Sphingomyelins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase