Viral replicon

Systemic viral vectors are restricted to certain parts of infected plants (usually the newly developing leaves, stem and roots), and are excluded from a significant part of plant biomass, including the mature leaves. Clearly, the creation of transgenic plants with a viral replicon precursor stably in-... 

Marillonnet, S., Thoetinger, C., Kandzia, R., Klimyuk, V., and Gleba, Y. (2005) Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat. Biotechnol, 23, 718-723. 

Hwang DR et al (2006) Synthesis and anti-viral activity of a series of sesquiterpene lactones and analogues in the subgenomic HCV replicon system. Bioorg Med Chem 14(1) 83—91... 

Other potent peptide mimetic NS3 protease inhibitors have been reported that incorporate a serine trap on the C-terminal end of the peptide. Thus, the inhibitory activity of telaprevir (VX-950, 59), (7nM vs. NS3, 300 nM vs. the la replicon) is based on truncation of the polypeptide substrate, maximizing binding by alteration of amino acids at the scissile site, and capping both N- and C-terminal ends, the latter with a known dicarbonyl serine trap. This compound has exhibited impressive antiviral activity in animals, and showed a 4.4 log drop in viral load in genotype 1-infected patients in a Phase lb clinical trial [110]. Telaprevir is expected to enter Phase 3 clinical trials in 2007. Additional bicyclo-proline-based P2 tetrapeptides, represented by analog 60 (Kj = 22 nM), have been explored. Although the compounds are selective inhibitors of NS3, little or no cell-based replicon activity was reported, presumably due to poor cellular permeability [111-114], A diastereomer of telaprevir, has been reported to inhibit the replicon with an EC50 of 0.55 pM [115]. 

Aiyar, A., Tyree, C. and Sugden, B. (1998) The plasmid replicon of EBV consists of multiple as-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element. EMBO J., 17, 6394-6403. 

Replicon DNA E1 expression vector Viral genome e2 expression vector... 

Cleave 10-200 pg cloned viral genome (or other replicon DNA) with appropriate enzyme to remove vector sequences. BamRl removes the pML2d vector from pl42-6 (Note 13). 

When using separate El and E2 expression plasmids and replicons, it is better to harvest DNA at shorter times after infection (2 and 4 d) because the viral genomes have a plasmid maintenance function that maintains them at a stable copy number compared to the expression vectors, which will be lost after several cell divisions. 

Marillonnet S, Giritch A, Gils M, et al. In planta engineering of viral RNA replicons efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. USA, 2004 101(18) 6852-6857. 

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