Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.
Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells.


CONCLUSIONS
We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.
Benedicto
Ignacio
I
Molecular Biology Unit, Hospital Universitario de la Princesa, Madrid, Spain.
Molina-Jiménez
Francisca
F
Barreiro
Olga
O
Maldonado-Rodríguez
Alejandra
A
Prieto
Jesús
J
Moreno-Otero
Ricardo
R
Aldabe
Rafael
R
López-Cabrera
Manuel
M
Majano
Pedro L
PL
D000998:Antiviral Agents / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D062445:Claudin-1; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020868:Gene Silencing; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology / Q000821:virology; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D019108:Tight Junctions / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
D000998:Antiviral Agents; C568108:CLDN1 protein, human; D062445:Claudin-1; D016898:Interferon-alpha; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D014759:Viral Envelope Proteins; D062826:Zonula Occludens-1 Protein; C091015:glycoprotein E2, Hepatitis C virus