Inhibition of hepatitis C virus NS3 function by antisense oligodeoxynucleotides and protease inhibitor.
Hepatitis C Virus (HCV) NS3 protease is an attractive target for antiviral agent development because it is required for viral replication. Because a stable cell culture system or small animal model to study HCV replication is not readily available, we constructed an in vitro model allowing the investigation of NS3 transcription, translation, and protease function. Sequences encoding for full length HCV genomes were cloned and transfected into HuH-7 human hepatocellular carcinoma cells to analyze NS3 transcription/translation. A plasmid pHCV ORF I luc that expresses the complete HCV coding region upstream of a luciferase reporter gene was designed to enable quantification of translated HCV proteins. Additionally, NS3 protease function was assessed by direct coexpression of NS3 and NS5 in HuH 7 cells, and the subsequent measurement of cleavage products. We found that antisense oligodeoxynucleotides (AS-ODN) interfered with NS3 translation in a dose dependent fashion; AS-ODN 5 cotransfection directed against NS3 sequences significantly inhibited protease activity as measured by cleaved NS5A levels. Finally, cleaved NS5A levels served as anindex of protease activity and Chymostatin, a protease inhibitor, almost completely blocked NS3 enzymatic activity. This cell culture system is useful in the assessment of potential antiviral agents on HCV NS3 expression and function.
Heintges
T
T
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Duesseldorf, Germany.
Encke
J
J
zu Putlitz
J
J
Wands
J R
JR
D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D016174:Hepacivirus / Q000187:drug effects*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020319:Oligodeoxyribonucleotides, Antisense; D011480:Protease Inhibitors; D012324:RNA-Dependent RNA Polymerase; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus