Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein.
Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.
Kang
Su-Min
SM
Department of Biotechnology, Yonsei University, Seoul, Korea.
Shin
Min-Jung
MJ
Kim
Jung-Hee
JH
Oh
Jong-Won
JW
D000818:Animals; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002417:Cattle; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D016174:Hepacivirus / Q000737:chemistry*; D022781:Hepatocytes / Q000473:pathology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007633:Keratins / Q000378:metabolism; D013058:Mass Spectrometry; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011233:Precipitin Tests; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism; D014758:Viral Core Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011506:Proteins; D011994:Recombinant Proteins; D014746:Vimentin; D014758:Viral Core Proteins; D007633:Keratins; D009532:Nickel; D014357:Trypsin; D009571:Nitrilotriacetic Acid