FIG. 2.

CLDN6 and CLDN9 mediate HCVpp entry. (A) 293T cells stably expressing claudins that were found in the Bel7402 cell line were infected with pseudotyped particles harboring VSV-G protein (black bar), envelope glycoproteins of HCV strain H77 (gray bars), and no env (white bars), with luciferase as a reporter (RLU, relative light units) (mean of n = 3) (error bars indicate SD). Naive 293T cells were used as a negative control. (B) Expression level of CLDN9 in Bel7402 cells treated with CLDN9-specific or irrelevant (irr.) shRNAs was determined by real-time PCR using CLDN9- and GAPDH-specific primers. The value of GAPDH was initialized to 1 (mean of n = 2) (error bars indicate SD). (C) Bel7402 cells treated with CLDN9-specific shRNAs were infected by pseudotyped particles bearing envelope glycoproteins as indicated in A. Results are presented as percentages of infection in irrelevant shRNA-treated cells (irr.) (mean of n = 3) (error bars indicate SD). (D) High homology between CLDN6 and CLDN9. The phylogenetic tree of EL1 of claudin family members was created by DNAMAN. The homology among EL1 of claudin family members is indicated as a percentage. (E) CLDN6 could also mediate HCVpp entry. Huh7, naive 293T, and 293T cells stably expressing individual CLDN1, CLDN6, CLDN9, CLDN2, CLDN3, CLDN4, CLDN7, CLDN11, CLDN12, CLDN15, CLDN17, and CLDN23 were infected with luciferase reporter viruses pseudotyped with envelope glycoproteins indicated as in A (RLU, relative light units) (mean of n = 3) (error bars indicate SD). (F) Flag-tagged CLDN9 could mediate HCV entry as efficiently as naive CLDN9. CLDN9-expressing and CLDN9-Flag-expressing 293T cells were infected by pseudotyped particles bearing envelope glycoproteins, as indicated in A, with luciferase as a reporter (mean of n = 3) (error bars indicate SD). The expression of CLDN9 and CLDN9-Flag was detected by Western blotting using antibodies against CLDN9 and Flag tag, with GAPDH as a control.