PARTS

This IRES (internal ribosome entry site) is derived from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation.

The sequence seen in Figure 1 is the one we used. This was cloned with a green fluorescent protein (iGEM part BBa_E0040) which we codon optimised for use in human cells and compared with a similar construct using EMCV IRES in place of the NKRF. We transfected the RNA of these constructs into Huh 7.5 cells in which, to our knowledge, the NKRF IRES has not been tested.

RNA Dependent RNA polymerase

RdRp is an enzyme which catalyses the replication of RNA from an RNA template, an essential protein for all viruses with an RNA genome. RdRp in Hepatitis C virus is also referred to as the NS5B protein, with a total molecular weight of 65 kDa. Heterelogous expression of NS5B has been achieved in insect and bacterial hosts, with RNA-dependent RNA synthesis initiated de novo (Behrens et al., 1996; Lohmann et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause insertion into the membrane, essential for HCV RNA replication (Moradpour et al., 2004). Amino acid substitution experiments of the 21 amino acid insertion sequence indicate functions beyond a membrane anchor role, with intracellular protein-protein interactions implicated (Moradpour et al., 2004).The C-terminal tail preceding the C-terminal hydrophobic insertion sequence interacts with structural elements including the β-hairpin loop of NS5b (Leveque et al., 2003). The β-hairpin loop inserts into the active site, believed to position the 3’ terminius of HCV viral RNA to initiate viral RNA synthesis (Hong et al., 2001). RdRp initiates viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002). NS5B activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al., 1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains ordered RNA structures, which are evolutionary conserved and contain crucial cis-acting elements for viral RNA replication. 150 nt in the 3’ termini of HCV RNA contains elements which are essential for RdRp binding and replication of viral RNA (Cheng et al., 1999; Yi and Lemon, 2003). We have designed an RdRp linked to an MS2 domain through a P2A linker, the MS2 domain is able to bind to the MS2 box and act in a negative-feedback loop to regulate RdRp expression in both ''E. coli'' and Huh 7.5 cells