title: Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B.
abstract: It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors.
  
  
  OBJECTIVE
  The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Lingbao
authors.initials: L
authors.lastname: Kong
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Fujimoto
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Haruyo
authors.initials: H
authors.lastname: Aoyagi
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Watashi
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Minetaro
authors.initials: M
authors.lastname: Arita
authors.affiliation: Department of Chemotherapy and Mycoses, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamagoe
authors.affiliation: RIKEN Center for Sustainable Resource Science, Saitama, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.affiliation: RIKEN Center for Sustainable Resource Science, Saitama, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Research Center for Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Sakamaki
authors.affiliation: Research Center for Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
authors.forename: Shizuko
authors.initials: S
authors.lastname: Ichinose
authors.affiliation: Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan aizaki@nih.go.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Aizaki
mesh_terms: D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000737:chemistry / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; C080107:NS4B protein, flavivirus; C118971:PREB protein, human; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins