Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus

Abstract

Hepatitis C virus (HCV) is a global challenge to public health. Several factors have been proven to be critical for HCV entry, including the newly identified claudin-1 (CLDN1). However, the mechanism of HCV entry is still obscure. Presently, among the 20 members of the claudin family identified in humans so far, CLDN1 has been the only member shown to be necessary for HCV entry. Recently, we discovered that Bel7402, an HCV-permissive cell line, does not express CLDN1 but expresses other members of claudin family. Among these claudins, CLDN9 was able to mediate HCV entry just as efficiently as CLDN1. We then examined if other members of the claudin family could mediate entry. We show that CLDN6 and CLDN9, but not CLDN2, CLDN3, CLDN4, CLDN7, CLDN11, CLDN12, CLDN15, CLDN17, and CLDN23, were able to mediate the entry of HCV into target cells. We found that CLDN6 and CLDN9 are expressed in the liver, the primary site of HCV replication. We also showed that CLDN6 and CLDN9, but not CLDN1, are expressed in peripheral blood mononuclear cells, an additional site of HCV replication. Through sequence comparison and mutagenesis studies, we show that residues N38 and V45 in the first extracellular loop (EL1) of CLDN9 are necessary for HCV entry.

FIG. 1.

Lack of expression of CLDN1 in the HCV-susceptible cell line Bel7402. (A) Cell tropism of HCVpp and its correlation to expression of CLDN1. Different cell lines were infected by HCVpp-bearing envelope glycoproteins of strain H77 (gray bar), and luciferase activity was measured as relative light units (RLU) 48 h postinfection. Pseudotyped particles containing VSV-G protein (black bars) or in the absence of envelope protein (white bars) were used as a control (mean of n = 3) (error bars indicate SD). Expression of CLDN1 in these cell lines was analyzed by semiquantitative RT-PCR and is shown under the bar graph, with GAPDH as a control. (B) Expression pattern of known human claudins in Bel7402 cells. The expression of known claudins in Bel7402 cells was analyzed by semiquantitative RT-PCR, using GAPDH as a control. −RT, control reaction of cDNA synthesis in which reverse transcriptase was not added.

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.

FIG. 3.

CLDN6 and CLDN9 mediate HCVcc infection. HCVcc infection was analyzed by real-time PCR. 293T cells expressing mock, CLDN1, CLDN6, or CLDN9 were challenged with HCVcc of strain J6/JFH. At 72 h postinfection, total RNAs were isolated from these cells and amplified with primers specific to the corresponding claudins or GAPDH. Huh7.5 cells were used as a positive control. The value of GAPDH was initialized to 1 (mean of n = 2) (error bars indicate SD).

FIG. 4.

Expression pattern of CLDN1, CLDN6, and CLDN9 in human liver, PBMCs, and human hepatocellular carcinoma cell lines. The cDNA samples of different sources were amplified by real-time PCR using CLDN1 (A)-, CLDN6 (B)-, CLDN9 (C)-, and GAPDH-specific primers. The value of GAPDH was initialized to 1 (mean of n = 2) (error bars indicate SD).

FIG. 5.

Key residues in EL1 of CLDN9 necessary for HCVpp entry. (A) Alignment of the N-terminal EL1 amino acid sequences of human CLDN1, CLDN3, CLDN6, and CLDN9 and murine CLDN1. Amino acid homology and differences are displayed by different shades of black. Number represents the positions of amino acids in full-length CLDN9 that were selected as candidates for further analysis. The numbers for known critical residues are shown in italics. (B and C) Wild-type and mutated claudin-transduced 293T cells were tested for susceptibility to HCVpp bearing envelope glycoproteins of strain H77 (gray bar) or VSV-Gpp (black bar) with luciferase as a reporter (RLU, relative light units) (mean of n = 3) (error bar indicate SD). (D and E) Expression of wild-type and mutated claudins was verified by real-time PCR. No signal was detected on naive 293T cells. The value of GAPDH was initialized to 1 (mean of n = 2) (error bars indicate SD).