环孢霉素A抑制乙型和丁型肝炎病毒进入通过亲环独立的干扰N

StephenW

Cyclosporin A inhibits Hepatitis B and Hepatitis D Virus entry by Cyclophilin-independent interference with the NTCP receptor

    Shirin Nkongoloa, 1,
    Yi Ni a, 1,
    Florian A. Lempp a,
    Christina Kaufman a, b,
    Thomas Lindner b,
    Katharina Esser-Nobis a,
    Volker Lohmann a,
    Walter Mier b,
    Stefan Mehrle a,
    Stephan Urban a, Corresponding author contact information, E-mail the corresponding author, E-mail the corresponding author

    a Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, D 69120 Heidelberg, Germany
    b Department of Nuclear Medicine, University Hospital Heidelberg, D 69120, Heidelberg, Germany


Abstract
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 8M. 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.
Abbreviations

    HBV, hepatitis B virus;
    HBsAg, hepatitis B surface antigen;
    HDV, hepatitis delta virus;
    NTCP, sodium taurocholate co-transporting polypeptide;
    TC, taurocholate;
    CsA, Cyclosporin A;
    HCV, hepatitis C virus;
    hNTCP, human sodium taurocholate co-transporting polypeptide;
    mNTCP, mouse sodium taurocholate co-transporting polypeptide;
    GFP, green fluorescent protein;
    PHH, primary human hepatocytes;
    DMSO, dimethyl sulfoxide;
    MyrB, Myrcludex B;
    HBeAg, hepatitis B virus early antigen;
    IF, immunofluorescence;
    PBS, phosphate-buffered saline;
    HBcAg, hepatitis B core antigen;
    DAPI, 4′,6-diamidin-2-phenylindol;
    MFI, mean geometric fluorescence intensity

StephenW

环孢霉素A通过抑制亲环独立的干扰乙型肝炎和丁型肝炎病毒进入与NTCP受体

    希林Nkongoloa ， 1 ，
    易倪一， 1 ，
    弗洛里安A. Lempp一个，
    克里斯蒂娜·考夫曼A，B ，
    托马斯·林德纳B，
    卡塔琳娜埃塞尔 - 诺比斯一个，
    沃尔克罗曼一，
    沃尔特·米尔B，
    斯特凡Mehrle一个，
    斯蒂芬城市一，通讯作者的联系信息，电子邮件通讯作者，电子邮件通讯作者

    传染病的部门，分子病毒学，大学医院海德堡，D 69120德国海德堡
    乙部门核医学，大学医院海德堡，D 69120 ，德国海德堡


摘要
背景及目的

慢性乙型肝炎和丁型肝炎是由人类乙型肝炎和丁型肝炎病毒的全球健康问题。大的包膜蛋白的肉豆蔻酰化前S1结构域介导的特异性结合到肝细胞中由牛磺胆酸钠共转运多肽（ NTCP ） 。 NTCP是已知由环孢菌素A所抑制本研究旨在刻画环孢素A对HBV / HDV感染的效果胆盐转运。
方法

HepaRG细胞，原代人肝细胞和易感NTCP表达肝癌细胞系已用于感染实验。环孢霉素A的作用方式进行了研究，通过比较不同的抑制剂，亲环素A / B / C沉默的细胞系以及NTCP变异和突变的效果。胆盐转运与HBV受体的功能是由牛磺胆酸钠的吸收和结合的HBVpreS定量研究。
结果

环孢霉素A抑制B型肝炎和D病毒感染过程中和 - 那么明显 - 前接种病毒。的HBVpreS到NTCP的结合阻断环孢素A浓度8M 。一个NTCP变种的HBVpreS约束力不足，但主管的胆盐运输表明电阻亲环素A / B / C没有废除转运体与受体的抑制作用，以环孢素A。沉默。与此相反，他克莫司，一个亲环独立钙调神经磷酸酶抑制剂，是无效的。
结论

通过牛磺胆酸钠共转运多肽HBV和HDV条目是由环孢菌素A的药物和病毒受体之间的相互作用是直接的，并与前S1结构域的功能性结合位点，其介导病毒进入重叠抑制。
缩略语

    乙型肝炎病毒，乙型肝炎病毒;
    乙肝表面抗原，乙肝表面抗原;
    HDV ，丁型肝炎病毒;
    NTCP ，牛磺胆酸钠共转运多肽;
    （TC），牛磺胆酸钠;
    环孢素，环孢霉素A ;
    丙型肝炎病毒，丙型肝炎病毒;
    hNTCP ，人牛磺胆酸钠共转运多肽;
    mNTCP ，鼠标牛磺胆酸钠共转运多肽;
    绿色荧光蛋白，绿色荧光蛋白;
    PHH ，原代人肝细胞;
    DMSO，二甲亚砜;
    MyrB ， Myrcludex乙;
    e抗原，乙型肝炎病毒早期抗原;
    中频，免疫荧光;
    PBS ，磷酸盐缓冲盐水;
    乙肝病毒核心抗原，乙型肝炎核心抗原;
    DAPI ， 4 '，6  - diamidin基-2  - 苯基吲;
    小额信贷机构，几何平均荧光强度