Characterization of a novel monocistronic reporter virus. (A) Schematic representation of full-length J6/JFH (top) and the reporter virus J6/JFH(p7-Rluc2A) (bottom). The J6/JFH(p7-Rluc2A) genome contains the coding sequence for Renilla luciferase followed by the FMDV (2A) peptide between p7 and NS2. RNA replication of J6/JFH(p7-Rluc2A) as determined by qRT-PCR (B) and luciferase assay (C) at 8, 24, 48, and 72 h posttransfection (white to black bars, respectively). For qRT-PCR analysis, HCV RNA copies were normalized to 50 ng of total RNA. (D) Infectious virus production of J6/JFH(p7-Rluc2A) at 8, 24, 48, and 72 h posttransfection (white to black bars, respectively). (E) Virus neutralization of J6/JFH(p7-Rluc2A). Virus-containing supernatants were treated with the isotype control antibody, α-IgG1, or the neutralizing antibody, α-CD81. The luciferase activity was determined at 48 h postinfection. The means and SEM of at least duplicate experiments are shown. GNN, J6/JFH(p7-Rluc2A)GNN; ΔE1E2, J6/JFH(p7-Rluc2A)ΔE1E2.

p7 functions at an early stage of virion morphogenesis. HCV core release into cell culture supernatants (A) and intracellular infectious virus accumulation (B) at 72 h posttransfection. HCV core release was determined by using a sensitive core-based ELISA (see Materials and Methods for details) on clarified cell culture supernatants harvested at 72 h posttransfection. For panel B, an analysis of infectious intracellular virus accumulation at 72 h posttransfection was performed. Lysates generated by multiple rounds of freeze-thaw were combined with isotype control antibody, α-IgGI (▪), or the HCV neutralizing antibody, α-CD81 (□), during infection of naive Huh-7.5 cells. Luciferase activity was determined at 48 h postinfection. The means and SEM of duplicate experiments are shown. GNN, J6/JFH(p7-Rluc2A)GNN; ΔE1E2, J6/JFH(p7-Rluc2A)ΔE1E2; Δp7, Δp7-Rluc2A.

Unprocessed forms of p7 are dispensable for infectious virus production. (A) Schematic representation of full-length monocistronic genome J6/JFH (top) and bicistronic genomes E2-IRES-p7 (middle) and p7-IRES-NS2 (bottom). The dotted line indicates the location of the EMCV IRES in bicistronic genomes. (B) Replication of mono- and bicistronic genomes at 8, 24, 48, and 72 h posttransfection (white to black bars, respectively) as determined by qRT-PCR. HCV RNA copies normalized to 50 ng of total RNA. (C) Infectious virus production of mono- and bicistronic genomes determined by TCID₅₀ assay at 24, 48, and 72 h posttransfection (light gray to black bars, respectively). The means and standard errors of the mean (SEM) of duplicate electroporations are shown. GNN, J6/JFH(GNN); ΔE1E2, J6/JFHΔE1E2.

p7 is essential for infectious virus production. (A) RNA replication of J6/JFH(p7-Rluc2A) genomes by luciferase assay at 8, 24, 48, and 72 h posttransfection (white to black bars, respectively). (B) Infectious virus production at 24, 48, and 72 h posttransfection (light gray to black bars, respectively). The deletions and mutations in the J6/JFH(p7-Rluc2A) genome are designated below. The positions of the point mutants are based on p7 numbering. (C) Infectious virus production at 72 h posttransfection of cells cotransfected with ΔE1E2 and Δp7 genomes (ΔE1E2+Δp7) and after one cell-free passage of the supernatant (ΔE1E2+Δp7[P1]). The means and SEM of duplicate experiments are shown. GNN, J6/JFH(p7-Rluc2A)GNN; ΔE1E2, J6/JFH(p7-Rluc2A)ΔE1E2; Δp7, Δp7-Rluc2A.

NS2, but not NS2-3, is required for infectious virus production. (A) Schematic representation of full-length monocistronic J6/JFH and bicistronic NS2-IRES-NS3 genomes. A dotted line indicates location of an EMCV IRES in NS2-IRES-NS3. (B) RNA replication of mono- and bicistronic genomes by qRT-PCR at 8, 24, 48, and 72 h posttransfection (white to black bars, respectively). HCV RNA copies normalized to 50 ng of total RNA. (C) Infectious virus production of mono- and bicistronic genomes as determined by TCID₅₀ assay at 24, 48, and 72 h posttransfection (light gray to black bars, respectively). (D) HCV core release of mono- and bicistronic genomes at 72 h posttransfection. GNN, J6/JFH(GNN); ΔE1E2, J6/JFHΔE1E2; ΔNS2, ΔNS2-IRES-NS3; NS2Δpro, NS2Δpro-IRES-NS3. (E) Intracellular infectious virus accumulation of reporter bicistronic genomes at 72 h posttransfection. Lysates generated by multiple rounds of freeze-thaw were combined with isotype control antibody, α-IgGI (▪), or the HCV neutralizing antibody, α-CD81 (□), during infection of naive Huh-7.5 cells. The luciferase activity was determined at 48 h postinfection. Gluc2AUbi, NS2-IRES-Gluc2AUbi; ΔE1E2, NS2-IRES-Gluc2AUbiΔE1E2; ΔNS2, ΔNS2-IRES-Gluc2AUbi; NS2Δpro, NS2Δpro-IRES-Gluc2AUbi. The means and SEM of duplicate experiments are shown.