Entire 3’ UTR is required for Core-binding to produce HCVtcp.

(A) Schematic representation of HCV genome and the regions used for the Core-RNA interaction assay. Stem-loops and colored lines depict in vitro synthesized and folded RNA fragments used. (B) In vitro interactions of RNA fragments with Core determined by AlphaScreen. Results for comparison among structure clusters across HCV genome (left) and among 3’ UTR and the fragments within the region (right) were obtained from two independent assays. (C) Trans-packaging of EmGFP RNA into HCV particles indicated by transduced RNA level in the inoculated cells (upper). Transduction of EmGFP RNA was determined at 12 hr post-inoculation. EmGFP RNA level in the co-transfected producer cells (lower) is shown. N: pCAG/NS3-5B, G: p/EmGFP, G3H: p/EmGFP-H3’UTR, encoding EmGFP followed with 3’ UTR of H77. G3J: p/EmGFP-J3’UTR, encoding EmGFP followed with 3’ UTR of JFH-1. cont; control with cells co-transfected with a pCAG-Neo empty vector, p/EmGFP-J3’UTR and pCAG/NS3-5B. (D) Entry of HCVtcp was blocked by anti-CD81 antibody (α-CD81), carried out as described in Fig 3C. Data were present as mean ± SEM, n = 4. (E) Comparison of tran-packaging of EmGFP RNAs, directed by 3’- or 5’ UTR. 5G3J: p/5’UTR-EmGFP-J3’UTR, encoding EmGFP flanked by 5’ UTR and 3’ UTR of JFH-1. 5G: p/5’UTR-EmGFP, addition of 5’ UTR at upstream of EmGFP. (C, D, E) Results shown represent the means of three independent experiments ± SEM. RNA copies are indicated as numbers per μg of total RNA.