Cell-specific Translation Initiation Activity Of HCV IRES And Its Mechanism

Objective:HCV 5'UTR contains an internal ribosome entry site (IRES), and its main functions are to regulate viral replication and to translate viral protein in cap-independent manner. In addition, the nucleotide sequence of HCV 5'UTR is highly conserved in different genotypes and strains, which makes it a good target for antiviral-drug research. The aim of this study was to analysis the differences of translation initiation activity of HCV 5'UTR with different deletions in different host cell lines and meanwhile to explore the mechanism.Methods:(1)Through the liposome-mediated gene-transfection technology, the Flue eukaryotic expression plasmid regulated by HCV IRES with different deletions and the Rluc eukaryotic expression plasmid into different cell lines. At 36 posttransfection:â‘ Total cellular RNA were harvested and detected by semiquantitative RT-PCR, and the pRL-TK plasmid was co-transfected as a nomalization cotrol;â‘¡The Flue gene's relative expression activity was detected through dual luciferase reporter gene assay system, and the different translation initiation activity was analysed in the different translation systems with different deletions of HCV IRES. (2)The plasmids with different deletions, which had significant difference in translational initiational activity, were selected, and those RNAs were in vitro transcripted to isolate specifically binding proteins.Results:(1)Between the activity of the full length HCV IRES and those with the deletion of the Domain I and the downstream single-stranded sequence, no significant difference was observed in Hela cells and C6 cells, however, the translational activity was decreased by 46% in L-02 cells and increased by 46% in 293T cells by the deletion. (2) Between the activity of the full length HCV IRES and those with the deletion of Domain II, in Hela cells, the activity was decreased to 51%, while in L-02 cells, C6 cells and 293T cells, it increased 40%,60% and 135% respectively. (3)The RNAs of HCV 5'UTR with different deletions were in vitro transcripted and the cellular proteins which can specifically bind to the RNAs were isolated. Conclusions:â‘ In L-02 cells, the Domain I of HCV 5'UTR could promote the initiation translation activity of HCV IRES, while it showed inhibitory effect in 293T cell lines and had no effect in C6 cells and Hela cells.â‘¡Domain II of HCV 5'UTR could enhance the translation initiation activity of HCV IRES in Hela cells, but inhibit it in the other cell lines.â‘¢Binding the in vitro transcribed RNA with the cellular protein by the streptavidin-biotin and magnetic beads, the specific binding protein were isolated. Still, no different proteins were observed among the different deletions of RNAs.