Trans-packaging system based on a replication-defective subgenomic replicon.

(A) Schematic representation of the HCV trans-packaging system. (B) Production of HCVtcp from replication-competent or replication-defective subgenomic replicon (WT or GND, respectively). Transfection with an empty vector pCAG-Neo and pHHSGR-JFH1/Gluc/GND was used to determine the background control (nc), unless described elsewhere. HCVtcp production was determined by quantification of the viral RNA in the transduced cells at 12 hr post-inoculation. The lower panel showed Gaussia luciferase (Gluc) activity released from producer cells. Core expression in producer cells was assessed by immunoblotting. (C) Blocking of HCVtcp entry by anti-CD81 antibody (left), and inoculation of Huh7-25 cells (right). Huh7.5.1 cells were pre-incubated with 20 μg/ml of anti-CD81 antibody (α-CD81) or mouse IgG (IgG) for 1 hr, followed by inoculation with GND HCVtcp. HCV RNA levels in the transduced cells (upper) and Gluc activity released from producer cells (lower) are shown. (D) Deletion of UTR impaired production of HCVtcp. Upper graph: Production of HCVtcp from GND and UTR deletion mutants. HCV RNA level in the transduced cells (upper graph) and producer cells (middle graph) were determined by qRT-PCR targeting NS5B region as shown in S1 Fig. Northern blot analysis of HCV RNA in Huh7.5.1 cells transfected with GND, Δ5’ UTR or Δ3’ UTR constructs (lower graph), Huh 7.5.1 cells were transfected with the mutant constructs and subjected to RNA extraction 72 hr post-transfection. 10 μg of total RNA was loaded to formaldehyde denaturing agarose gel electrophoresis and followed by Northern hybridization; a DIG-labeled RNA probe targeting to NS5B was used. 28S rRNA was used to demonstrate equal loading. Comparable Core expression in the producer cells was determined by western blotting. (B, C, D) Results of HCV RNA in transduced and producer cells, and reporter activity in producer cells represent the means of three independent experiments ± SEM. HCV RNA copies are indicated as numbers per μg of total RNA for each assay, and Gluc activities are indicated as RLU per μl.