Figure 4: HCV pseudo-particles (HCVpp) with envelope proteins of SB strain infected B lymphocytes, but not hepatic cells.

(a) Claudin1 (CLDN1) or Occuludin (OCLN) expression confers weak susceptibility of Raji cells to JFH1 infection (Upper panel). Immunoblot analysis demonstrated that Claudin-1 and Occludin proteins were expressed by transfection of plasmids. (b) The respective expression vectors of CLDN1 and OCLN were transfected into both Raji and Huh7.5.1 cells. Luciferase-encoding pseudo-particles bearing the indicated envelope proteins were used to infect recipient Raji cells transfected with control vector (white), CLDN1-expressing vector (grey), or OCLN-expressing vector, or control Huh7.5.1 cells (black). For HCVpp, the respective E1E2 genes are indicated in parentheses. Luciferase assay was performed at 3 days after transduction (*P<0.05, t-test). Values are normalized to the RLU background measured in mock-infected cells (mean of n=3; error bars, s.d.). RLU: relative luminescence units. (c) The respective expression vectors of CLDN1 and OCLN were transfected and their protein expression levels were confirmed in both Raji and Huh7.5.1 cells. (Left panel) Expression of both CLDN1 and OCLN (tight junction molecules) promoted HCVpp(SB) entry (right panel) (*P<0.05, t-test, n=3). Error bars represent s.d. (d) Cells were transfected to express shRNA against CLDN1 or OCLN and examined for silencing effects by immunoblots. shRNAs against DNA polymerase ζ (an irrelevant shRNA for HCV entry) were used as the negative and positive controls, respectively. Two different shRNA clones targeting either CLDN1 or OCLN were used. (e) Silencing of tight junction molecules CLDN1 and OCLN did not inhibit HCVpp(SB) entry in lymphocytes while expression of CLDN1 and OCLN is required for the susceptibility to HCVpp(JFH1) in hepatocytes. Cells were challenged with HCVpp (black bars: pseudotyped with SB or JFH1 E1E2) or MLV-Gpp (white bars) encoding a luciferase reporter (*P<0.05, t-test, n=3). Error bars represent s.d.