title: Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B.
abstract: The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Itsuki
authors.initials: I
authors.lastname: Hamamoto
authors.forename: Yorihiro
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Toru
authors.initials: T
authors.lastname: Okamoto
authors.forename: Hideki
authors.initials: H
authors.lastname: Aizaki
authors.forename: Minyi
authors.initials: M
authors.lastname: Liu
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Mori
authors.forename: Takayuki
authors.initials: T
authors.lastname: Abe
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Michael M C
authors.initials: MM
authors.lastname: Lai
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Miyamura
authors.forename: Kohji
authors.initials: K
authors.lastname: Moriishi
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
mesh_terms: D000595:Amino Acid Sequence; D001921:Brain; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D051605:Kv Channel-Interacting Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D002135:Calcium-Binding Proteins; D051605:Kv Channel-Interacting Proteins; D017361:Viral Nonstructural Proteins; C496750:KCNIP1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase