Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection

Abstract

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.

FIG. 1.

Sequence alignments of human, tupaia, and mouse CD81 LEL, claudin 1 EL1, and occludin EL2.

FIG. 2.

Tupaia CD81 binds HCV E2. (A) TRX-CD81 LEL fusion proteins were expressed and purified. Equal amounts of the fusion proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue or immunoblotted with the anti-CD81 MAbs JS81, 5A6, and 1.3.3.22. Lane 1, TRX-mouse CD81 LEL; lane 2, TRX-human CD81 LEL; lane 3, TRX-tupaia CD81 LEL. MW, molecular weight (in thousands). (B) The interaction between HCV E2 and TRX-CD81 LEL fusion proteins was analyzed by EIA. Values are the means ± standard deviations of three independent experiments. O.D., optical density. (C) CHO cells were infected with lentiviruses containing human or tupaia CD81 sequences or mock lentivirus. Three days later, the cells were probed with the anti-CD81 MAb 5A6 (left) or polyclonal antibodies (pAb) (right) and analyzed for CD81 expression by flow cytometry. (D) HCV E2 binding to CD81-transduced CHO cells was detected with anti-E2 polyclonal antibodies by flow cytometry. In panels C and D, the values on the y axis indicate counts, and those on the x axis CD81 expression. The experiment was repeated at least three times, and one representative result is shown.

FIG. 3.

HCV E2 binds tupaia SR-BI. (A) CHO cells were infected with lentiviruses containing human or tupaia SR-BI sequences or mock lentivirus. Three days later, SR-BI expression was identified by immunoblotting with an anti-SR-BI MAb. MW, molecular weight (in thousands). (B) SR-BI- or mock-transduced CHO cells were incubated with the E2 extract, and E2 binding was assayed by flow cytometry using anti-E2 polyclonal antibodies. The experiment was repeated at least three times, and one representative result is shown. (C) CHO cells described in the legend to panel A were incubated with anti-SR-BI polyclonal serum collected from mouse that received DNA immunization with human or tupaia SR-BI expression plasmid or with preimmune control serum and analyzed for cell surface SR-BI expression by flow cytometry. In panels B and C, the values on the y axis indicate counts, and those on the x axis E2 or SR-BI expression, respectively.

FIG. 4.

Tupaia CD81 supports HCVpp infection. (A) HepG2 cells were infected with lentiviruses containing human or tupaia CD81 sequences or mock lentivirus. Three days later, the cells were probed with the anti-CD81 MAb 5A6 (left) or polyclonal antibodies (right), and CD81 expression was assessed by flow cytometry. The values on the y axis indicate counts, and those on the x axis CD81 expression. (B) Human or tupaia CD81- or mock-transduced HepG2 cells were incubated with anti-CD81 MAbs (5A6, JS81, and 1.3.3.22; 5 μg/ml), polyclonal antibodies (50 μg/ml), or mouse IgG1 (5 μg/ml) for 1 h prior to the addition of HCVpp or the control (no E1E2). At 72 h postinfection, the cells were harvested, and the level of luciferase activity (relative light units [RLU]) was determined. The values are the means ± standard deviations of results from three independent experiments.

FIG. 5.

Tupaia CLDN1 supports HCVpp infection. (A) 293T cells were infected with lentiviruses containing human or tupaia CLDN1 sequences or mock lentivirus, and the expression of CLDN1 was assayed by immunoblotting. MW, molecular weight (in thousands). (B) Transduced 293T cells were infected with HCVpp or control (no E1E2). Three days later, HCVpp infection was determined by measuring the level of luciferase activity. The values are the means ± standard deviations of three independent experiments.

FIG. 6.

Tupaia OCLN facilitates the HCVpp infection of mouse NIH 3T3 cells. (A) NIH 3T3 cells were infected with lentiviruses containing human or tupaia OCLN sequences or mock lentivirus. Three days later, OCLN expression was assayed by immunoblotting with an anti-human OCLN MAb. MW, molecular weight (in thousands). (B) NIH 3T3 cells were infected with several combinations of lentiviruses and then with HCVpp. Three days later, the HCVpp infection levels were determined by measuring cellular luciferase activity. Naïve Huh7.5 and NIH 3T3 cells were used as positive and negative controls, respectively. H, human; T, tupaia; chimera, human-tupaia chimeric OCLN, in which the EL2 of humans was replaced by that of tupaia; T-ΔEL2, tupaia OCLN lacking EL2. The values are the means ± standard deviations of three independent experiments.

FIG. 7.

PTHs can be infected by HCVpp. (A) HCVpp of diverse genotypes and control (no E1E2) were used to infect PTHs; Huh7.5 cells were used as a positive control. Three days after infection, the cells were lysed and assayed for luciferase activity. The values are the means ± standard deviations of three independent experiments. (B) Inhibition of HCV infection by anti-CD81 antibodies. PTHs and Huh7.5 cells were incubated with the human anti-CD81 MAbs 5A6, JS81, and 1.3.3.22, mouse IgG1 at 5 μg/ml, polyclonal antibodies at 50 μg/ml, human or tupaia SR-BI immune serum, or preimmune control serum at 1:50 dilution for 1 h prior to the addition of HCVpp. The values are the means ± standard deviations of three independent experiments.

FIG. 8.

PTHs can be infected by HCVcc. (A) The PTHs, Huh7.5 or HepG2 cells were incubated with J6/JFH-1 chimera HCVcc for 5 h, the media were removed, and the wells were washed five times with the culture medium. Fresh medium was added at 500 μl per well, and the plate was placed in a 5% CO₂-humidified incubator at 37°C. The medium was changed every 24 h. The HCV RNA level in supernatants collected at days 0 to 8 was determined by real-time quantitative RT-PCR using a commercial kit (PG Biotech, Shenzhen, China). The supernatant at day 0 refers to the medium recovered from the cell culture at 3 h after washing the well and replacing the medium. (B) Culture medium from PTHs infected with HCVcc was collected every day for a total of 8 days and was used to infect freshly seeded Huh7.5 cells. Three days later, new HCV protein expression was detected by immunofluorescence analysis. The experiment was repeated at least three times, and one representative result is shown. (C) After detection by immunofluorescence analysis, the stained foci were counted in quadruplicate wells, and the mean infectious titers (FFU/ml) of culture medium collected from HCVcc-infected PTHs, Huh7.5, and HepG2 cells were calculated.