Hepatitis C virus NS5A protein down-regulates the expression of spindle gene Aspm through PKR-p38 signaling pathway
Journal
Journal of Biological Chemistry
Journal Volume
283
Journal Issue
43
Pages
29396-29404
Date Issued
2008
Author(s)
Abstract
Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G2/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G2/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein downregulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma. ? 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
SDGs
Other Subjects
Amines; Amino acids; Chromosomes; Fluid dynamics; Gene expression; Laws and legislation; Nucleic acids; Oligonucleotides; Organic acids; Viruses; Affymetrix; Amino acid substitutions; Aneuploidy; Cell cycles; Cultured cells; Gene expression profiles; Hepatitis C viruses; Hepatocellular carcinomata; Hepatocytes; In vitro; In-vivo; Laser capture microdissection; Mitotic cells; Mitotic spindles; Molecular mechanisms; Mouse models; Nonstructural proteins; Oligonucleotide microarrays; Overexpression; Persistent infections; Promoter activities; Real time polymerase chain reactions; Regulatory effects; Signaling pathways; Western blots; Real time systems; abnormal spindle like microcephaly; cell cycle protein; messenger RNA; nonstructural protein 5A; unclassified drug; amino acid substitution; aneuploidy; animal cell; animal cell culture; animal experiment; article; Aspm gene; Bagg albino mouse; cell cycle arrest; cell cycle G2 phase; cell cycle M phase; chromosomal instability; DNA transfection; down regulation; gene expression; Hepatitis C virus; hydrodynamics; laser capture microdissection; liver cell; liver cell carcinoma; male; mitosis spindle; mouse; nonhuman; priority journal; protein expression; protein isolation; transcription regulation; virus gene; virus genome; Western blotting; Animals; Cercopithecus aethiops; COS Cells; Down-Regulation; eIF-2 Kinase; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Viral; Humans; Mice; Models, Biological; Nerve Tissue Proteins; NIH 3T3 Cells; p38 Mitogen-Activated Protein Kinases; Viral Nonstructural Proteins; Hepatitis C virus
Type
journal article