Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir

Direct-acting antivirals (DAAs) effectively eradicate chronic hepatitis C virus (HCV) infection, although HCV genotype 3a is less responsive to these drugs. We aimed to develop genotype 3a infectious cultures and study the effects of inhibitors of NS5A and NS5B and resistance to sofosbuvir-the only nucleotide analog approved for treatment of chronic HCV infection.The developed HCV genotype 3a full-length genome (DBN3a), with a strain-DBN coding sequence, modified NS5B consensus sequence, pS52 untranslated regions, and coding mutations from a culture-efficient JFH1-based core-NS5A (DBN) recombinant, was transfected into Huh7.5 cells. The efficacy of selected DAAs was determined in dose-response assays, in which the number of HCV-infected cells was measured after incubation with different concentrations of the specific DAA. Long-term culture of infected Huh7.5 cells with increasing concentrations of sofosbuvir was used to promote selection of HCV-resistant variants.We engineered a DBN3a variant with 17 substitutions (DBN3acc) that had replication and propagation kinetics in Huh7.5 cells comparable with prototype J6/JFH1. The adaptive mutations also produced culture-efficient DBN-based recombinants with NS5B from HCV genotype 3a strains S52 and DH11. Compared with genotype 1a, genotype 3a was less sensitive to daclatasvir, ledipasvir, and elbasvir, but equally sensitive to ombitasvir, velpatasvir, beclabuvir, dasabuvir, MK-3682, and sofosbuvir. Exposure of Huh7.5 cells infected with DBN3a to sofosbuvir led to identification of an escape variant with substitutions in NS5B, including the resistance-associated substitution S282T. This variant showed increased infectivity of Huh7.5 cells, compared with DBN3a, and was genetically stable in cell cultures without sofosbuvir. Sofosbuvir, MK-3682, dasabuvir, or combinations of sofosbuvir and ledipasvir or sofosbuvir and velpatasvir had decreased efficacy against infection with the DBN3a sofosbuvir escape variant.We developed a system for highly efficient culture of HCV genotype 3a. Genotype 1a has a high genetic barrier to resistance for sofosbuvir, whereas resistance to this DAA can be induced in genotype 3a. We therefore isolated HCV genotype 3a variants with reduced sensitivity to sofosbuvir, with increased fitness and with cross-resistance to other NS5B inhibitors. These findings indicate that sofosbuvir escape variants could compromise the effectiveness of nucleotide analogs against HCV. GenBank accession numbers: KX280712-KX280716.