Replicon

Stuyver LJ, McBrayer TR, Tharnish PM, Hassan AE, Chu CK, Pankiewicz KW, Watanabe KA, Schinazi RF, Otto MJ (2003a) Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabohtes, J Virol 77 10689-10694... 

Stuyver LJ, McBrayer TR, Tharnish PM, Clark J, Hollecker L, Lostia S, Nachman T, Grier J, Bennett MA, Xie MY, Schinazi RF, Morrey JD, Inlander JL, Furman PA, Otto MJ (2006a) Inhibition of hepatitis C replicon RNA synthesis by beta-D-2 -deoxy-2 -fluoro-2 -C-methylcytidine a specific inhibitor of hepatitis C virus replication, Antivir Chem Chemother 17 79-87... 

A major limitation in the development of anti-HCV compounds was the lack of a virus replication system. This was finally overcome with the development of a novel replicon system that directed persistent replication in a cell culture format (Lohmann et al. 1999). Using such a system, it was possible to demonstrate antiviral activity of an NS3/4A inhibitor in a cell culture assay, and demonstrate potency on par with treatment with interferon-a (Pause et al. 2003). 

Resistance to NS3/4A inhibitors was primarily tested using the replicon system in tissue culture and closely parallels what was seen with resistance to HIV-1 PI. 

IFN-y has potent activity against HCV in the subgenomic replicon system (Dash et al. 2005 Frese et al. 2002 Lanford et al. 2003). Synergistic immunomodulatory effects of IFN-ylb and IFN-a have been reported (Wang et al. 2006). However, a pilot study of IFN-y at a dose of 100-400pg three times per week showed no antiviral efficacy in patients infected with HCV genotype 1 who had not responded to standard therapy or who had relapsed (Soza et al. 2005). 

Currently, there is no approved antiviral therapy specifically targeting hepatitis C virus (HCV). The development of an HCV replicon system and our improved understanding of the structure and function of HCV proteins have led to the development of several classes of specific HCV inhibitors. NS3-4A protease inhibitors and NS5B polymerase inhibitors are furthest in development as discussed in Chaps. 2-4 (De and Migliaccio 2005 Manns et al. 2007 Pawlotsky et al. 2007). 

The development of resistance against HCV NS3/4 protease inhibitors will become a major challenge for the clinical use of these new compounds. Clinical trials of telaprevir (VX-950) have shown that mutations at different positions are rapidly selected (Sarrazin et al. 2005). In vitro studies indicate that cells bearing repUcons with those mutations are associated with different levels of resistance to telaprevir (< 10-fold change to >40-fold change in sensitivity). However, telaprevir-resistant mutants remain susceptible to interferon-a, at least in the replicon system. Likewise, replicon mutants that are resistant to boceprevir are still sensitive to interferon-a (Tong et al. 2006). 

Data represent the mean from 4- 6 tubers per line (Ig tissue per trial 3 replicons). Western blot analyses were positive. 

The evolutionary history of symbiotic nitrogen fixers is therefore a tale of coevolution, which occurred in the shadow of their hosts, chasing their growing roots, and striving for adaptation. It is an example of how bacterial genetics has managed to keep pace with the creative power of eukaryotic sexual recombination. Mobile replicons, insertion elements, and symbiotic islands prone to move have helped rhizobia to succeed in their pursuit. The race, naturally, is not over and, looking at it from a distance, what we have. seen, compared to what we have yet to see, is probably just a cloud of dust. 

Fig. 2.1 (A) Structure of the HCV genome, with 5 - and 3 -untranslated regions and individual proteins of the polyprotein indicated Regions of the genome (approximately 3,000 bases) are drawn to scale. Structural protein regions are in light gray, nonstructural proteins in white, and untranslated regions in dark gray. (B) Structure of a typical replicon sequence, with the antibiotic resistance gene (Neo ) in place of the structural region and the second IRES (EMCV) inserted. The NS2 sequence is often rwt present in the replicon.

Western blot has also been reported [62, 63]. These assays have been widely used over the last several years but have recently been largely supplanted by the subgenomic replicon. 

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... 

Denis-Larose, C. Bergeron, H. Labbe, D., et al., Characterization of the Basic Replicon of Rhodococcus Plasmid pSOX and Development of a Rhodococcus Escherichia Coli Shuttle Vector. Applied and Environmental Microbiology, 1998. 64(11) pp. 4363-4367. 

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