Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.
BACKGROUND
Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis.


OBJECTIVE
To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation.


METHODS
A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection.


RESULTS
Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity.


CONCLUSIONS
Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.
Oem
Jae-Ku
JK
Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, 120 Veterinary Road, Saskatoon, Sask., Canada S7N 5E3.
Xiang
Zhonghua
Z
Zhou
Yan
Y
Babiuk
Lorne A
LA
Liu
Qiang
Q
D020121:5' Untranslated Regions; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012318:RNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D012367:RNA, Viral; D008156:Luciferases; D012318:RNA Polymerase I