The effects of the conserved extreme 3' end sequence of hepatitis C virus (HCV) RNA on the in vitro stabilization and translation of the HCV RNA genome.
OBJECTIVE
The discovery of an additional 98-base in the extreme 3' end of the hepatitis C virus (HCV) has fueled much speculation as to the role of this sequence on the behavior of the virus. It is now known that this additional 98-base sequence is present and conserved amongst HCV genotypes. This sequence is capable of forming complex and stable high-order structures that may be important in stabilizing the RNA to degradation, facilitating translation and regulating replication of the virus. We have examined the possible role of the HCV extreme 3' end sequence in stabilizing the HCV RNA genome and regulating translation in vitro.


METHODS
The extreme 3' end sequence was cloned to downstream of two pre-existing two HCV clones: HCV1 (genotype 1a) and HCV-BK (genotype 1b). The reconstructed full-length clones were then tested in vitro for their stability and translation efficiency.


RESULTS
We showed that the addition of the conserved 3' end sequence greatly enhanced the stability of HCV1 RNA but had only minimal effect on HCV-BK RNA in mammalian cytoplasmic extracts, suggesting that the requirements for HCV RNA stability vary amongst isolates. Following the optimization of in vitro translation conditions, it was demonstrated that the addition of this 3' end sequence did not affect the translation level from either HCV clone.


CONCLUSIONS
The conserved 3' end of the HCV genome confers differential stabilizing effects on two HCV genotype 1 isolates and has no obvious role in the in vitro translation of either clone.
Fang
J W
JW
Department of Pediatrics, University of Florida, Gainesville, USA. jfang@ICNpharm.com
Moyer
R W
RW
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D008322:Mammals; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics
D020413:3' Untranslated Regions; D012367:RNA, Viral