Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP)

Abstract

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug-resistant viruses highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection-susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having a median inhibitory concentration <0.2 μM.

Conclusion: This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform.

Copyright © 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases.

Figures

Figure 1

Cyclosporin A (CsA) blocked HBV infection. (A) Schematic representation of the schedule for exposing HepaRG cells to compounds and HBV. HepaRG cells were pretreated with compounds for 2 hours and then inoculated with HBV for 16 hours. After washing out the free HBV and compounds, the cells were cultured with the medium in the absence of compounds for an additional 12 days to quantify HBs protein secreted from the infected cells into the medium. Black and dotted bars indicate the interval for treatment and without treatment, respectively. (B) CsA 4 μM, heparin 25 U/mL, bafilomycin A1 200 nM, lamivudine 1 μM, anti‐FLAG 10 μg/mL, and anti‐HBs antibody 10 μg/mL, were tested for effect on HBV infection according to the protocol shown in (A). (C‐E) HBc protein (C), HBV DNAs, and cccDNA (D) in the cells as well as HBe antigen in the medium (E) at 12 days postinfection according to the protocol shown in (A) were detected by immunofluorescence, real‐time PCR analysis, southern blot, and ELISA. Red and blue in (C) show the detection of HBc protein and nuclear staining, respectively. (F) PHHs were treated with the indicated compounds and infected with HBV using the protocol shown in (A). The levels of HBV DNAs in the cells, as well as of HBs and HBe antigens in the medium, were determined. Statistical significance was determined using the Student t test (*P < 0.05, **P < 0.01).

Figure 2

CsA reduced internalized HBV. (A) HepaRG cells were treated with or without various concentrations of CsA (0.5, 1, 2, and 4 μM) as shown in Fig. 1A. HBV infection was monitored by HBs and HBe secretion. (B) HBV DNA in core particles was detected by southern blot analysis of DNA extracts from HepAD38 cells treated for 6 days with or without tetracycline 0.5 μg/mL and lamivudine 1 μM. (C) Upper scheme indicates the treatment schedule of HepaRG cells with compounds and HBV. HepaRG cells were infected with HBV for 16 hours. After washing out the input virus, cells were cultured in the absence of compounds for 8 days. The cells were then cultured with compounds (lamivudine 1 μM, entecavir 1 μM, IFN‐α 100 IU/mL, or heparin 25 U/mL) for 4 days and recovered for detection of HBV DNA. Black and dotted boxes indicate the periods with and without treatment, respectively. Lower graph shows the quantified relative HBV DNA level in cells treated according to the above scheme. (D) Upper scheme shows the experimental procedure for examining the attached and internalized HBV. (a) The cells were pretreated with compounds (heparin 25 U/mL, lamivudine 1 μM, or CsA 4 μM) at 4°C for 2 hours and then treated together with HBV at 4°C for 3 hours to allow HBV attachment to the cells. After washing out the free virus, cell surface HBV DNA was extracted and quantified by real‐time PCR. (b) After attachment of HBV at 4°C for 3 hours and the following wash, the cells were cultured in the presence or absence of compounds at 37°C for 16 hours to allow the cells to internalize bound HBV. The cells were then trypsinized and extensively washed prior to quantifying the cellular HBV DNA. The lower graphs show the level of HBV DNA attached to the cells (a) and internalized inside the cells (b). “Background” in (b) indicates the signal from cells incubated at 4°C, instead of 37°C, for 16 hours after washing out the virus in (b), which shows the background signal level of the assay. (E) The upper scheme shows the procedure for the time of addition experiment. Compounds (CsA 4 μM, anti‐HBs antibody 10 μg/mL, or heparin 25 U/mL) were applied beginning 2 hours prior to HBV infection (a), beginning during HBV infection (b), or beginning immediately after HBV infection (c) until 24 hours postinfection. HBs and HBe protein secretion were measured at 12 days postinfection. Middle and lower graphs indicate HBs and HBe secretion, respectively, from the cells treated according to the above scheme. (F) Preincubation of HBV with compounds. HBV was preincubated with the indicated compounds for 30 minutes at 37°C. Compounds were then removed by ultrafiltration. The recovered compound‐treated HBV was used to infect HepaRG cells (16 hours incubation), and HBV infection was monitored with HBs antigen secreted into the medium at 12 days postinfection. *P < 0.05, **P < 0.01, N.S., not significant.

Figure 3

CsA showed a pan‐genotypic anti‐HBV effect. (A) PHHs were treated with compounds (CsA 4 μM or heparin 25 U/mL) according to the scheme in Fig. 1A with different genotypes of HBV inoculum, and either HBs protein in the medium or HBV DNA in the cells at 12 days postinfection was quantified. (B) CsA did not affect the entry of HCV. Huh‐7.5.1 cells were pretreated with the indicated compounds for 1 hour and then infected with HCVpp for 4 hours. At 72 hours postinfection, intracellular luciferase activity was measured. *P < 0.05, **P < 0.01, N.S., not significant.

Figure 4

Effect of immunosuppressants on HBV infection. (A,C) HepaRG cells were treated with or without the indicated compounds at 2 μM (FK506 4 μM) in (A), and CsA (2 and 4 μM) and PSC833 (2 and 4 μM) in (C), according to the scheme in Fig. 1A. HBs (A,C) and HBe (C) secretion was determined. (B) Effect of compounds on the activity of the calcineurin/NF‐AT pathway. Jurkat cells transfected with pNF‐AT‐luc and pRL‐TK were stimulated with PMA and ionomycin in the presence or absence of CsA, FK506, and PSC833 for 24 hours to measure the luciferase activity. (D) Cyclophilin binding activity of CsA, FK506, and PSC833 was determined in a competitive binding assay as described in the Materials and Methods using a CsA‐derived fluorescent probe. IC50s (μM) for the inhibition of probe binding to CyPA, CyPB, and CyPD are shown. *P < 0.05, **P < 0.01.

Figure 5

NTCP inhibitors blocked HBV infection. (A) NTCP transporter activity was examined following CsA, FK506, rapamycin, and PSC833 treatment of 293 cells overexpressing NTCP, as described in the Materials and Methods. Dose‐response curves and IC50s for inhibition of NTCP transporter activity are shown. (B) NTCP transporter activity was measured in HepG2‐NTCP cells treated with or without CsA 10 μM or tauroursodeoxycholic acid (TUDCA) 10 μM as a positive control. (C) Expression of mRNAs for NTCP, CyPA, CyPB, and GAPDH in HepaRG, PHHs, HepG2, Huh‐7, HeLa, and FLC4 cells was determined by RT‐PCR. (D) HepaRG cells were treated with or without CsA 4 μM, ursodeoxycholate 100 μM, cholic acid 100 μM, propranolol 100 μM, progesterone 40 μM, bosentan 100 μM, and heparin 25 U/mL according to the scheme in Fig. 1A. Secretion of HBs and HBe was quantified. (E) HepG2 cells overexpressing NTCP (HepG2‐NTCP) and the parental HepG2 cells were pretreated with or without CsA or heparin for 2 hours, then treated with HBV for 16 hours. HBV infection was monitored with HBs and HBe secreted from the cells. (F) AlphaScreen assay to evaluate the binding between NTCP and large envelope protein (LHBs) as described in the Materials and Methods. (a) Left, His‐tagged GST (white bars) or NTCP (black bars) are incubated with large (LHBs), middle (MHBs), or small envelope protein (SHBs). Right, His‐tagged NTCP and other nonrelevant proteins, LCK and FYN, and GST were incubated with LHBs, MHBs, and GST. (b‐e) His‐tagged GST (white bars) or NTCP (black bars) were incubated with LHBs in the presence of varying amounts of pre‐S1 lipopeptide HBVpreS/2‐48myr (b; 0, 7.7, 15.3, 30.7, and 61.3 μM), CsA (c; 0, 37.5, 75, 150, and 300 μM), FK506 (d; 31, 63, 125, 250, and 500 μM), and SCYX1454139 (e; 0, 37.5, 75, 150, and 300 μM), respectively. *P < 0.05, **P < 0.01.

Figure 6

Analysis of CsA analogs. (A,B) Anti‐HBV activity of CsA analogs. HepaRG cells were treated with or without dimethyl sulfoxide (DMSO), heparin 10 U/mL, lamivudine 1 μM, CsA 4 μM, or its analogs, SCYX618806, SCYX1774198, SCYX827830, and SCYX1454139 (A) or alisporivir (B) at 4 μM, as shown in Fig. 1A to measure HBs and HBe secretion level. (C) Chemical structures of CsA and its derivatives. (D) Dose‐response curves for CsA analogs. HepaRG cells were treated with or without various concentrations of SCYX618806, SCYX827830, or SCYX1454139 (0.25, 0.5, 1, 2, and 4 μM) as shown in Fig. 1A. (E) IC50s (μM) for CsA and its analogs in blocking HBV infection are shown. CC50s (μM) determined by the MTT cell viability assay are also shown. (F) PHHs were treated with CsA and its derivatives at 4 μM or left untreated according to the protocol in Fig. 1A, and HBV infection was monitored by HBs protein secretion. *P < 0.05, **P < 0.01.