Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-α.
OBJECTIVE
Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α.


METHODS
Huh7.5 hepatoma cells were transfected with RNA of genotype 1-7 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays.


RESULTS
Cells infected with viable HCV that expressed NS5A of genotypes 1-7 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC(50)) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC(50) values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region.


CONCLUSIONS
We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 1-7 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.
Copenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark.
Troels K H
TK
Scheel
Judith M
JM
Gottwein
Lotte S
LS
Mikkelsen
Tanja B
TB
Jensen
Jens
J
Bukh
D000998:Antiviral Agents / Q000494:pharmacology*; D002219:Carbamates; D045744:Cell Line, Tumor; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D057166:High-Throughput Screening Assays; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D016898:Interferon-alpha / Q000494:pharmacology*; D009154:Mutation; D010641:Phenotype; D011759:Pyrrolidines; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013997:Time Factors; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D000998:Antiviral Agents; D002219:Carbamates; D004791:Enzyme Inhibitors; D007093:Imidazoles; D016898:Interferon-alpha; D011759:Pyrrolidines; D011994:Recombinant Proteins; D014633:Valine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir