Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors.
OBJECTIVE
Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome alpha-subunit 7 (PSMA7) or Hu antigen R (HuR).


METHODS
In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells.


RESULTS
PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells.


CONCLUSIONS
A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.
Korf
Mortimer
M
Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
Jarczak
Dominik
D
Beger
Carmela
C
Manns
Michael P
MP
Krüger
Martin
M
D000954:Antigens, Surface / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000187:drug effects* / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005814:Genes, Viral / Q000187:drug effects; D016679:Genome, Viral; D016174:Hepacivirus / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000378:metabolism / Q000821:virology; D010957:Plasmids; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis / Q000187:drug effects*; D021122:Protein Subunits / Q000037:antagonists & inhibitors* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000235:genetics; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D000954:Antigens, Surface; D003546:Cysteine Endopeptidases; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D034741:RNA, Small Interfering; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; C098770:ELAVL1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C488662:PSMA7 protein, human