Zinc mesoporphyrin induces rapid proteasomal degradation of hepatitis C nonstructural 5A protein in human hepatoma cells.
OBJECTIVE
The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) plays a critical role in HCV replication and is an attractive target for the therapy of HCV infection. So far, little is known about the posttranslational regulation of NS5A protein and its precise role in HCV RNA replication. Our objectives were to elucidate the down-regulation of NS5A protein and HCV RNA replication by zinc mesoporphyrin (ZnMP) and the mechanism by which this process occurs.


METHODS
Human hepatoma cells expressing HCV proteins were used to investigate the posttranslational regulation of ZnMP on NS5A protein by Western blots and immunoprecipitation. Real-time quantitative reverse transcription polymerase chain reaction was used to determine the effects of ZnMP on HCV RNA replication.


RESULTS
ZnMP selectively and markedly down-regulated NS5A protein levels by increasing degradation of NS5A protein (half-life fell from 18.7 hours to 2.7 hours). The proteasome inhibitors epoxomicin and MG132 significantly abrogated degradation of NS5A protein by ZnMP without affecting levels of NS5A in the absence of ZnMP. Analysis of immunoprecipitates with an antiubiquitin antibody revealed polyubiquitination of NS5A, suggesting that ZnMP induces ubiquitination of NS5A protein. In addition, 10 micromol/L of ZnMP reduced HCV replication by approximately 63% in the Con1 replicon cells, approximately 70% in J6/Japanese fulminant hepatitis 1 HCV-transfected cells, and approximately 90% in J6/Japanese fulminant hepatitis 1 HCV-infected cells without affecting cell viability.


CONCLUSIONS
ZnMP produces a rapid and profound down-regulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent catabolism. ZnMP may hold promise as a novel agent to treat HCV infection.
Hou
Weihong
W
Liver-Biliary-Pancreatic Center and the Liver, Digestive Disease and Metabolism Laboratory, Carolinas Medical Center, Charlotte, North Carolina 28232-2861, USA. weihong.hou@carolinashealthcare.org
Tian
Qing
Q
Zheng
Jianyu
J
Bonkovsky
Herbert L
HL
D000998:Antiviral Agents / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D006207:Half-Life; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D008665:Metalloporphyrins / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D055550:Protein Stability; D012367:RNA, Viral / Q000096:biosynthesis; D013997:Time Factors; D014162:Transfection; D054875:Ubiquitination; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D000998:Antiviral Agents; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008665:Metalloporphyrins; D009842:Oligopeptides; D046988:Proteasome Endopeptidase Complex; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C058069:zinc mesoporphyrin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin