Hcv genotype 4 replicons
A genotype and residue technology, applied in the field of preparation and use of the replicon, can solve problems such as not yet produced, genotype 4 replicon not described, etc.
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[0095] Preparation method of genotype 4 replicon
[0096] As shown in Example 1, following recognition of the HCV genotype 4 replicon, introduction of relevant adaptive mutations into the corresponding genotype HCV RNA can confer RNA replication competence, especially in mammalian cells in vitro. Therefore, the present invention provides a method for improving the replication ability of genotype 4HCV viral RNA in eukaryotic cells, comprising one or more of the following:
[0097] (a) Substitution of residue 200 of NS3 with glutamic acid,
[0098] (b) Substitution of residue 343 of NS3 with lysine or arginine,
[0099] (c) Substitution of residue 511 of NS3 with arginine,
[0100] (d) Substitution of residue 34 of NS4A with lysine or arginine,
[0101] (e) substitution of residue 52 of NS4A with valine, or
[0102] (f) Substitution of residue 179 of NS5A with proline. In one aspect, the method includes at least two substitutions in (a)-(f).
[0103] In any of the above meth...
Embodiment 1
[0118] Example 1: Generation of a Robust Genotype 4 Hepatitis C Virus Subgenomic Replicon
[0119] This example shows that adaptive mutations are identified from genotype 4 HCV viral replicons capable of replicating in Huh7 cells, and that HCV replicons with these adaptive mutations are useful tools for antiviral drug screening.
[0120] Materials and methods
[0121] cell culture
[0122] Three HCV permissive cell lines were used in these studies: Huh7-Lunet, 51C and 1C. Huh7-Lunet was obtained from ReBLikon GmbH (Mainz, Germany) (Friebe et al., J Virol 79:380-92 (2005)). The source of 51C cells, stable genotype 1a H77 and genotype 1b Con-1Rluc-Neo replicon cells has been described previously (see Robinson et al. Antimicrob Agents Chemother 54:3099-106 (2010)). 1C cells were obtained by curing the GS-5885 resistant genotype 1a replicon clone derived from 51C cells (supra). Among the 50 clones screened, this clone showed the highest permissiveness for GT1a and 1b replicons...
Embodiment 2
[0165] Example 2 Screening for new HCV inhibitors of genotype 4
[0166] Example 1 shows that agents known to be HCV inhibitors of other genotypes, such as genotype 1, can be tested for their utility against genotype 4 HCV using the genotype 4 replicon. It is also envisaged that agents not yet known to inhibit HCV could also be screened using the genotype 4 replicon.
[0167] Candidate HCV inhibitors may be small molecule drugs, peptides or proteins such as antibodies, or may be nucleic acid based such as siRNA. The candidate HCV inhibitor is incubated with cells containing the genotype 4 replicon at a suitable temperature for a period of time to allow the replicon to replicate in the cells. The replicon may include a reporter gene such as luciferase, and in this case, at the end of the incubation period, the cells are assayed for luciferase activity as an indicator of the level of replication. Luciferase expression can be quantified using a commercial luciferase assay. Alt...
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