Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories
Hossam Gewaid, Haruyo Aoyagi, Minetaro Arita, Koichi Watashi, Ryosuke Suzuki, Shota Sakai, Keigo Kumagai, Toshiyuki Yamaji, Masayoshi Fukasawa, Fumihiro Kato, Takayuki Hishiki, Ayako Mimata, Yuriko Sakamaki, Shizuko Ichinose, Kentaro Hanada, Masamichi Muramatsu, Takaji Wakita, Hideki Aizaki
Abstract
Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.