Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor

doi:10.1016/j.jhep.2013.11.022

Journal of Hepatology

Volume 60, Issue 4, April 2014, Pages 723-731

https://doi.org/10.1016/j.jhep.2013.11.022 Get rights and content

Background & Aims

Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection.

Methods

HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding.

Results

Cyclosporin A inhibited hepatitis B and D virus infections during and – less pronounced – prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8 μM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive.

Conclusions

HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry.

Introduction

Hepatitis B is one of the major global infectious diseases, initiated by the human hepatitis B virus (HBV). It causes acute and chronic infection of the liver, giving rise to cirrhosis and hepatocellular carcinoma. Current treatment options for chronic HBV infections (interferon-alpha, nucleoside/nucleotide analogues) rarely result in clearance of the virus. Despite the availability of vaccines, the prevalence of hepatitis B surface antigen (HBsAg) carriers is only slowly dropping, particularly in developing countries [1].

HBV is a small, enveloped DNA virus belonging to the family of hepadnaviridae. It shows pronounced liver tropism and distinct species specificity. Hepatitis D virus (HDV) is an RNA virus taking advantage of the HBV envelope proteins for assembly, secretion and also hepatocyte-specific entry. In vitro infection systems were limited for a long time and host factors serving as bona fide receptors for virus entry have not been functionally characterized until recently [2]. Nevertheless, it was demonstrated that the myristoylated preS1 region of the HBV L-protein mediates specific virus-host cell interaction [3]. Minor changes in this region abrogate infectivity of the respective virus mutants [4], [5], [6]. A functional HBV/HDV receptor was recently identified [7]: sodium taurocholate co-transporting polypeptide (NTCP) is a sodium-dependent bile salts transporter exclusively expressed in the basolateral membrane of differentiated hepatocytes. Its physiological substrates are taurocholate (TC) and derivatives thereof, preferentially in a conjugated form [8]. Several cholestasis-inducing drugs including cyclosporin A (CsA) inhibit NTCP [9], [10], [11]. CsA is a cyclic peptide consisting of eleven amino acids. It is used as an immunosuppressive drug, e.g., after organ transplantation or for treatment of autoimmune diseases. CsA exerts its immunosuppressive activity by binding to cyclophilins and consequently inhibiting calcineurin. Structural CsA analogues (e.g., alisporivir or NIM811) also bind to cyclophilins, but are not immunosuppressive. Tacrolimus (FK506) is an immunosuppressive drug, structurally different from CsA. It inhibits calcineurin in a cyclophilin-independent manner. Apart from its immunosuppressive activity, CsA and some non-immunosuppressive analogues interfere with hepatitis C virus (HCV) replication through binding to cyclophilin, which is a host-dependency factor of HCV replication [12], [13].

In this study, we used established [14] culture systems (PHH, HepaRG) as well as the recently developed, NTCP-based infection systems [7], [15] to investigate the direct effect of CsA on HBV entry through NTCP.

Section snippets

Plasmids

hNTCP (human) cDNA (Origene) was subcloned into the lentiviral vector pWPI-puro, which co-expresses puromycin resistance. mNTCP (mouse) cDNA was obtained from Stephan Herzig. hNTCP-m81-178 chimera and the hNTCP-KG157-158GR mutant were generated by overlapping PCR. All NTCP variants were introduced into the green fluorescent protein (GFP) co-expressing lentiviral vector pWPI-GFP. HBV genome was cloned into a cytomegaloviral vector (pcDNA3.1/Zeo(−) HBV 1.1) for HBV transfection. For cyclophilin

Inhibition of HBV and HDV infection by CsA

CsA has been previously shown to inhibit the transport of several substrates (e.g., TC, cholate) of NTCP [9], [10], [11]. Since NTCP was recently identified as a bona fide receptor for HBV and HDV, we investigated whether CsA might also interfere with HBV infection. Cytotoxicity of CsA under all conditions used could be excluded by trypan blue cell viability tests (Supplementary Fig. 1). PHH, differentiated HepaRG cells as well as hNTCP-overexpressing HuH7 and HepG2 cells were infected with HBV

Discussion

In this study we demonstrate that CsA inhibits HBV and HDV infection by directly interfering with NTCP-mediated binding and entry into host cells. We used the authentic systems PHH and HepaRG cells and took advantage of other cell lines that gained HBV susceptibility by NTCP overexpression.

CsA interferes with HCV replication in a cyclophilin-dependent manner [12], [13]. CsA was also described to indirectly influence HBV replication by binding to cyclophilins and therefore interfering with

Financial support

This work received funding by the Deutsche Forschungsgemeinschaft (DFG) UR72/7-1, FOR1202/UR72/5-1 and the Deutsche Zentrum für Infektionsforschung (DZIF) to SU and the Hartmut Hoffmann Berling International Graduate School HBIGS to SN.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgements

We thank Thomas Weiss for providing PHHs and the Charitable Foundation Human Tissue and Cell Research (HTCR), Regensburg, for making human tissue available for research. We are grateful to Christa Kuhn for providing antibodies. We thank Vanesa Madan for providing CsA and cells for initial experiments.

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^†: These authors contributed equally to this work.

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Research ArticleCyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor

Background & Aims

Methods

Results

Conclusions

Introduction

Section snippets

Plasmids

Inhibition of HBV and HDV infection by CsA

Discussion

Financial support

Conflict of interest

Acknowledgements

Virology

Gastroenterology

Biochem Pharmacol

Hepatology

Methods

Biochem Pharmacol

J Hepatol

Virology

Genome Biol

Curr Top Membr

Biochim Biophys Acta

Gastroenterology

Hepatology

Hepatitis B virus biology

Microbiol Mol Biol

Viral and cellular determinants involved in hepadnaviral entry

World J Gastroenterol

Myristoylated preS1-domain of the hepatitis B virus l-protein mediates specific binding to differentiated hepatocytes

Hepatology

Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-s1 domain

J Virol

Infectivity determinants of the hepatitis B virus pre-s domain are confined to the n-terminal 75 amino acid residues

J Virol

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

Elife

Research Article
Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor