Team:Warwick/Parts

Below is an interactive schematic of our system, hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results.

5' Promoter

This is derived from the 5i UTR of the HCV virus strain 1b isolate Con1. The secondary structure of this sequence acts as a binding site and initiates replication of the system by RdRp. It also includes the first 16 amino acids of the first gene of HCV as this has been shown to increase the efficacy of binding of the RdRp to the 5' promoter.

IRES

This acts as an initiation for eukaryotic ribosomes and begins translation of the following protein sequence. We compared two different IRESs: the classical EMCV IRES used in many papers,which has been shown to be compatible with replicons and the NKRF IRES derived from the 3’UTR of the mammalian NF-kappaB repressing factor, which we also chose to test because during investigation regarding the efficacy and strength of the EMCV IRES, the NKRF derived IRES was shown to be 30-fold more efficient however had never been previously used in Huh7.

MS2 Box

The MS2 box acts as a binding site for the MS2 coat protein, formed downstream in the replicon, which represses translation hence preventing exponential growth of the replicon.

Neomycin Resistance

This was included in order to select for the cells which had been successfully transfected with the replicon. This is commonly used in human cell studies and allows survival of eukaryotic cells in the prescence of geneticin.

Aptazyme

This is an RNA enzyme which self- cleaves in the presence of theophylline. Theophylline is not endogenous to mammalian cells hence acts as a selective “off-switch” in an instance such as, Hepatitis C infection or tumour formation which is exacerbated by lack of DPP-IV.

siRNA

This is the functional element of our replicon and shows a potential area for RNA experimentation. This sequence was specifically designed using RNA fold and investigating the minimum free energy bond formation, in order to form a secondary structure in the positive sense that would not include a length of double stranded RNA longer than 16 bases and hence would not be recognised by Dicer and our replicon would remain intact for further replication. However, in the negative sense the secondary structure will form a hairpin recognised by Dicer which will result in cleavage and release of the siRNA from the RNA strand allowing it to bind in a complimentary fashion to the 3’UTR of DPP-IV mRNA and cause RNA silencing hence reducing the amount of DPP-IV protein present in the cell. We used siRNA sequences from various papers to design this hairpin.

MS2 Coat Protein

This coding sequence forms a protein which binds to the MS2 box, hairpin type structure, and represses translation of the replicon.

P2A

This is derived from the Porcine Teschovirus-1 genome and cleaves with high efficiency and leaving no scar following the formation of a polyprotein MS2 coat protein, P2A and RdRp. This is required as the HCV genome which has one open reading frame from which one long polyprotein is produced. RdRp is the final protein in this polygenic mRNA and hence does not have a start codon, adding a start codon could be disastrous for the function of RdRp.

RdRp

This is a polymerase derived from Hepatitis C Virus Strain 1b isolate Con1 which catalyses the replication of RNA from an RNA template, reffered to as NS5B in the contact of the HCV genome. 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 with other intracellular protein-protein interactions implicated (Moradpour et al., 2004) making the final 63 bases essential for HCV RNA replication in eukaryotic cells (Moradpour et al., 2004). In prokaryotic cells the final 21 amino acids are expendable. 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).

3' Promoter

Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative promoter sequences previously identified by Heinz et. Al. as templates for replication by the HCV RdRP.