title: Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection.
abstract: Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis.
  
  
  
  TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Si C
authors.initials: SC
authors.lastname: Tran
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Tu M
authors.initials: TM
authors.lastname: Pham
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Lam N
authors.initials: LN
authors.lastname: Nguyen
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.|Korea National Institute of Health, Cheongwon-gun, South Korea.
authors.forename: Eun-Mee
authors.initials: EM
authors.lastname: Park
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Yun-Sook
authors.initials: YS
authors.lastname: Lim
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea sbhwang@hallym.ac.kr.
authors.forename: Soon B
authors.initials: SB
authors.lastname: Hwang
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D016174:Hepacivirus / Q000502:physiology*; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000502:physiology; D053586:Virus Internalization; D014779:Virus Replication / Q000502:physiology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D018797:Cell Cycle Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C474831:TRIB3 protein, human; C494918:AKT1 protein, human