Selection and Characterization of HCV Replicon Cells That Are Resistant to Cyclosporine A and Temperature Shift In Vitro

The hepatitis C virus (HCV) is a serious health threat globally. Current therapies are not tolerated well, have a low response rate, and there is no available vaccine. New viral targets for drugs are urgently needed. The aim of this study was to characterize resistance to chemical and non-chemical treatments of the HCV replicon and determine a viral target for new treatment options. Replicon cells were treated with both Cyclosporine A (CsA) and temperature shift treatment (39°C). Resistant replicon cells were attained by double treatment with selection antibiotics and anti-viral treatments, in concert with live cell sorting techniques. Resistant cell lines were analyzed and RNA was extracted. This RNA was electroporated into naïve or cured cells, creating new cell lines. These new cell lines were then tested for resistance. Resistant replicon RNA was also sequenced and compared to non-resistant strains. After cell lines had been attained with high levels of resistance, and RNA was electroporated into naïve or cured replicon cells, these new cell lines also showed resistance. This indicated that the viral RNA was the source of the treatment resistance. There were unique mutations at the amino acid level in both CsA and temperature shift resistant cell lines. These unique mutations in both the CsA and temperature shift resistant replicon genomes indicate changes in the NS5B and NS3 proteins, respectively. Further work on the protein structures with the amino acid substitutions and their interactions could lead to new targets for therapies in patients.