期刊
JOURNAL OF VIROLOGY
卷 85, 期 17, 页码 8913-8928出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.00049-11
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资金
- Copenhagen University Hospital, Hvidovre
- Proof of Concept Foundation
- Lundbeck Foundation
- Novo Nordisk Foundation
- Danish Council for Independent Research-Medical Sciences
- A. P. Moller and the Chastine Mc-Kinney Moller Foundation
- Danish Cancer Society
- Faculty of Health Sciences, University of Copenhagen
- Ghent University [01G00507]
- Belgian state (IUAP) [P6/36-HEPRO]
- Research Foundation-Flanders [31500910]
To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Delta 40) or 25-aa (Delta 25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Delta 40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Delta 40 or Delta 25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP Delta 40 acquired various deletions in EGFP, while 2a(J6)Delta 40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A Delta 40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.
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