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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) (⌬40) or 25-aa (⌬25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. ⌬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 ⌬40 or ⌬25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP⌬40 acquired various deletions in EGFP, while 2a(J6)⌬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⌬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.Hepatitis C virus (HCV) is a small, enveloped virus classified as a member of the family Flaviviridae. Its single-stranded RNA genome contains one long open reading frame (ORF) encoding structural proteins (core and envelope glycoproteins E1 and E2), p7, forming a putative ion channel, and nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (8). The NS5A phosphoprotein consists of 3 domains (47). While NS5A domains I and II are important for replication (46,47), domain III has been shown to be important for viral assembly (5,28,45). Due to significant genetic heterogeneity, HCV was classified into 7 major genotypes and numerous subtypes that differ in ϳ30% and ϳ20% of their sequences, respectively (42). Genotypes also differ biologically (34) and in their rates of response to interferon (IFN)-based combination therapy and specific antivirals (27, 36).HCV poses a major socioeconomic burden, with approximately 180 million chronically infected individuals worldwide who are at risk of developing liver cirrhosis and hepatocellular carcinoma (15, 41). Treatment options are limited; combination therapy with IFN-alfa-2 and ri...