Recombinant hepatitis C virus genotype 5a infectious cell culture systems expressing minimal JFH1 NS5B sequences permit polymerase inhibitor studies

• Published in: Virology (New York, N.Y.) Vol. 522; pp. 177 - 192
• Main Authors: Humes, Daryl, Ramirez, Santseharay, Jensen, Tanja B., Li, Yi-Ping, Gottwein, Judith M., Bukh, Jens
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2018
• Subjects: Antiviral Agents - isolation & purification; Antiviral Agents - pharmacology; Cell Culture Techniques - methods; Cell Line; DAA; Direct acting antivirals; Drug Evaluation, Preclinical - methods; Genotype; Genotype 5a; HCV; Hepacivirus - classification; Hepacivirus - genetics; Hepacivirus - growth & development; Hepatitis C virus; Hepatocytes - virology; Humans; Infectious cell-culture; Liver Cancer; Polymerase inhibitors; Replication; Viral Nonstructural Proteins - genetics; Virus Cultivation - methods; aa; ORF; Genotype 5a; DAA; E; Infectious cell-culture; nt; NNI; Liver Cancer; FFU/ml; UTR; Replication; Polymerase inhibitors; HCV; NI; Direct acting antivirals; Hepatitis C virus
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/j.virol.2018.05.020

The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.