Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner.

Leiyun Weng (1) , Yuichi Hirata (2) , Masaaki Arai (3) , Michinori Kohara (2) , Takaji Wakita (4) , Koichi Watashi (4, 5) , Kunitada Shimotohno (5, 6) , Ying He (1) , Jin Zhong (1) , Tetsuya Toyoda (1)

1 LMVI - Key Laboratory of Molecular Virology & Immunology
2 Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Biology
3 Pharmacology Laboratory, Pharmacology Department V, Mitsubishi Tanabe Pharma Corporation
4 Department of Virology II
5 Laboratory of Human Tumor Viruses, Department of Viral Oncology, Institute for Virus Research
6 CIT - Chiba Institute of Technology

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.

Leiyun Weng, Yuichi Hirata, Masaaki Arai, Michinori Kohara, Takaji Wakita, et al.. Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner.. Journal of Virology, 2010, 84 (22), pp.11761-70. ⟨10.1128/JVI.00638-10⟩. ⟨pasteur-00624936⟩