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  • Since its development the HCV replicon system


    Since its development, the HCV replicon system (Lohmann et al., 1999) has been a critical tool in the HCV antiviral discovery process (Gottwein et al., 2009, Imhof and Simmonds, 2010, Lohmann et al., 1999, Pietschmann et al., 2001, Imhof and Simmonds, 2010, Gottwein et al., 2009). The replicon assay can be performed in stable cell lines or using transient KPT-185 systems (Ismail et al., 2013, Qi et al., 2009). However, despite its utility, there are some technical drawbacks to using this system. Creation of new mutant replicons takes significant amounts of time, with weeks needed to develop new transient replicons and months needed for selection and characterization of stable replicons. In addition, some mutations cannot be studied in the context of a stable or transient replicon assay due to insufficient replication. HCV NS3 protease activity and antiviral inhibition can also be studied in vitro using purified NS3 protease domain or full length NS3/4A protein. However, individual protein expression and purification is time consuming and can become a large burden when working with a large number of constructs. In this study, we introduce a fast and simple assay to study HCV NS3/4A activity and inhibition by PIs using protein expressed in Escherichia coli cells. Full-length NS3/4A was cloned into a pET SUMO expression vector; NS3/4A protein was expressed using IPTG induction, and the resulting whole-cell lysates were used for analysis of protease activity using an assay that takes less than 30min. Assay conditions including protein expression, lysis buffer and assay buffer conditions, and amount of lysate used were optimized, resulting in IC50 values that were highly reproducible. In addition, we show that this assay system is effective for characterization of mutants, population and clonal phenotyping of patient samples, and analysis of NS3/4A activity in multiple genotypes.
    Materials and methods
    Discussion and conclusions The HCV replicon assay has revolutionized HCV research and drug development. Stable replicon assays and transient replicon constructs produce reliable, reproducible EC50 data that are an essential component of bringing greatly needed new therapies through development (Lanford et al., 2003, Peng et al., 2013, Vrolijk et al., 2003, Wong et al., 2012). Unfortunately, the replicon system cannot currently fill every research need. Some HCV mutations observed in vivo have significant low replication capacity and thus cannot be studied using the replicon system. Specifically, replicon chimeras encoding sequences from patient viral isolates can be especially prone to lack of replication. Furthermore, replicon assays are time-consuming. In this study, we introduce a fast and simple assay to study HCV NS3 protease activity and drug inhibition by using E. coli cell lysates. Full-length NS3 and NS4A were cloned into a pET SUMO expression vector; SUMO-NS3/4A protein expression was induced for 4h by IPTG, and the whole cell lysate was used for the assay. We optimized assay conditions, including protein expression, lysis buffer and assay buffer conditions, and the amount of lysate to be used in the assay. The IC50 values from the assay were highly reproducible even when using previously frozen lysate, allowing for the convenience of large lysate batch production to be utilized. In addition, the assay itself takes less than 30min to run, allowing for fast, efficient production of data. Analysis of NS3/4 mutations previously characterized in the replicon using this method demonstrated that fold-changes in IC50 values observed for mutants in this assay were comparable to fold-changes in EC50 values generated from the replicon assay, thus making it amenable to the characterization of mutations observed during the nonclinical and clinical development processes. Notably, similar correlations between the IC50 and EC50 fold-change values for two different PI classes were observed. Telaprevir is a reversible covalent inhibitor of NS3/4A, while GS-9451 and GS-9256 are both non-covalent NS3/4A inhibitors (Chatel-Chaix et al., 2010, Dvory-Sobol et al., 2012d) that select variant at positions R155 and D168 (Wong et al., 2010, Wong et al., 2011). Consistency of results between two different drug PI subclasses suggests that this assay will not be limited by the mechanism of action of the PI being tested and may be widely applied to any PI being investigated.