Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication.
Hepatitis C virus (HCV), a hepacivirus member of the Flaviviridae family, has a positive-stranded RNA genome, which consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5' and 3' ends. The 5'NTR was found to contain an internal ribosomal entry site (IRES), which is required for the translation of HCV mRNA. Moreover, the 5'NTR is likely to play a key role in the replication of viral RNA. To identify the cis-acting element required for viral RNA replication, chimeric subgenomic replicons of HCV were generated. Dissection of the replication element from the translation element was accomplished by inserting the polioviral IRES between the serially deleted 5'NTR of HCV and ORF encoding neomycin phosphotransferase. The deletions of the 5'NTR of HCV were performed according to the secondary structure of HCV. Replicons containing domains I and II supported RNA replication and further deletion toward the 5' end abolished replication. The addition of domain III and the pseudoknot structure of the 5'NTR to domains I and II augmented the colony-forming efficiency of replicons by 100-fold. This indicates that domains I and II are necessary and sufficient for replication of RNA and that almost all of the 5'NTR is required for efficient RNA replication.
Kim
Yoon Ki
YK
Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San31, Hyoja-Dong, Pohang, Kyungbuk 790-784, Korea.
Kim
Chon Saeng
CS
Lee
Song Hee
SH
Jang
Sung Key
SK
D020121:5' Untranslated Regions; D001483:Base Sequence; D015153:Blotting, Western; D017353:Gene Deletion; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D019868:Kanamycin Kinase / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000096:biosynthesis* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D012313:RNA; D019868:Kanamycin Kinase