Internal ribosome entry site within hepatitis C virus RNA.
The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA. However, a capped methylated RNA showed a much higher activity as mRNA than did the capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES). Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES. The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.
Tsukiyama-Kohara
K
K
Department of Microbiology, Tokyo Metropolitan Institute of Medical Science, Japan.
Iizuka
N
N
Kohara
M
M
Nomoto
A
A
D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins