title: Hepatitis C virus entry is impaired by claudin-1 downregulation in diacylglycerol acyltransferase-1-deficient cells.
abstract: Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV.
  
  
  OBJECTIVE
  In this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4α and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression.
authors.affiliation: Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea.
authors.forename: Pil Soo
authors.initials: PS
authors.lastname: Sung
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Murayama
authors.affiliation: Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea.
authors.forename: Wonseok
authors.initials: W
authors.lastname: Kang
authors.affiliation: Graduate School of Biomedical Science and Engineering, College of Medicine, Hanyang University, Seoul, Republic of Korea.
authors.forename: Myung-Sun
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
authors.forename: Seung Kew
authors.initials: SK
authors.lastname: Yoon
authors.affiliation: Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Fukasawa
authors.affiliation: Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, and Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Graduate School of Biomedical Science and Engineering, College of Medicine, Hanyang University, Seoul, Republic of Korea.
authors.forename: Hyongbum
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
authors.affiliation: Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea ecshin@kaist.ac.kr.
authors.forename: Eui-Cheol
authors.initials: EC
authors.lastname: Shin
mesh_terms: D045744:Cell Line, Tumor; D062445:Claudin-1 / Q000235:genetics*; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics* / Q000472:pathogenicity*; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D053586:Virus Internalization
chemical_list: D062445:Claudin-1; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; C494881:DGAT1 protein, human; D051048:Diacylglycerol O-Acyltransferase