Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening

Abstract

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo r marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. Approximately 170 million people globally test positive for hepatitis C virus (HCV) (9, 11). Infection by HCV results in a high degree of chronic hepatitis. In addition to inducing liver damage, a significant proportion of these infections also result in hepatocellular carcinoma. Although current treatments for hepatitis caused by HCV include interferon in combination with ribavirin (18), approximately 50 to 60% of individuals still are not able to resolve infection (15). Therefore, there is an unmet medical need to develop more effective therapies to treat HCV infection. Until 1999, all cell-based screening efforts for HCV drug discovery relied on surrogate viral systems, such as bovine viral diarrhea virus, and the potential development of assays where activities of specific viral targets could be monitored. In 1999, a significant breakthrough in studying HCV RNA replication occurred when the Bartenschlager laboratory developed the HCV replicon system, a tissue culture system that faithfully mimicked all of the RNA replication events of the HCV life cycle (21). This initiated a phase of intensified research into the mechanisms of HCV RNA translation, replication, and protein processing. It also ushered in a new era for HCV drug discovery, since it was now possible to test the effects of inhibitors of traditional targets, such as NS3 protease, helicase, and NS5B polymerase, in an authentic, in vitro HCV RNA replication system (1).The original replicon system (21) was constructed by replacement of genes from the HCV genome that are not essential for HCV RNA replication, e.g., the structural genes, p7 and NS2, with a genetic cassette carrying an antibiotic resistance gene and the internal ribosomal entry site (IRES) from encephalomyocarditis virus (EMCV). This resulted in the formation of a dicistronic, selectable, subgenomic HCV replicon (2-4, 21) whose replication requires RNA elements in both nontranslated regions as well as the nonstructural proteins, including NS3 protease, helicase, and polymerase. Therefore, the HCV replicon system can be used for identifying inhibitors against all of these components (1).Cell-based screening efforts in a high-throughput format to identify novel inhibitors and viral or host targets have recently been described (6,23,34). The first ...

Development of Intergenotypic Chimeric Replicons To Determine the Broad-Spectrum Antiviral Activities of Hepatitis C Virus Polymerase Inhibitors

“…All stable replicon lines were cultured using the same conditions plus the addition of 250 g/ml G418 (Invitrogen). The GT1b Con-1 reporter replicon construct, pBB7M4 hRLuc, was constructed as described previously (12). The pBS.KS plasmid was purchased from Stratagene (La Jolla, CA).…”

“…The resulting product was cloned into the appropriate pBS.KS shuttle vector at the EcoR1 and StuI (or SnaBI) sites. The chimeric sequence, along with the Con-1 3Ј UTR and the HDV ribozyme, was subcloned from the pBS.KS shuttle vector to the subgenomic Con-1 replicon, pBB7M4 hRLuc, at the unique EcoR1 and SpeI restriction sites (12).…”

“…The NS5B polymerase sequences were isolated from patient samples infected with GT2b, -3a, -4a, -5a, or -6a and used to replace the corresponding sequence in a highly active GT1b Con-1 reporter replicon system (12). Due to a significant reduction in the replication fitness of these chimeric replicons in transient assays, stable cell lines were created from the replication-competent intergenotypic chimeric replicons for use in susceptibility assays.…”

In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

“…To improve its replication efficiency, the S2204I adaptive mutation in BB7 was reverted to the wt amino acid while three different adaptive mutations, E1202G, T1280I, and S2197P, were introduced into BB7 to construct the BB7M4 replicon (9). The replicon was further modified to include a humanized Renilla luciferase (hRLuc) gene and the autocleavage site of the foot-and-mouth disease virus 2A protease so that HCV RNA replication could be monitored by a reporter assay (9). The reporter replicon construct was electroporated into Huh7 cells to generate a reporter replicon cell line, DSR.…”

Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a

“…The incorporation of reporter genes, such as secreted alkaline phosphatase (SEAP), beta-lactamase, or firefly luciferase, facilitated antiviral screening efforts by increasing assay throughput and improving assay statistics (e.g., signal-tonoise ratio and Z factor) (16,24,36). The humanized Renilla reniformis luciferase gene (hRluc) is being increasingly utilized in the HCV infectious virus system due to its enhanced signalto-noise ratio and decreased gene length (12,15,23). More recently, a humanized Gaussia princeps luciferase gene (hGluc) with even higher substrate turnover rates and smaller gene size than hRluc has been described (32).…”