乙型肝炎病毒感染高效地经由人类NTCP非粘附肝癌细胞

StephenW

Article | Open
Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide

    Kaori Okuyama-Dobashi, Hirotake Kasai, Tomohisa Tanaka, Atsuya Yamashita, Jun Yasumoto, Wenjia Chen, Toru Okamoto, Shinya Maekawa, Koichi Watashi, Takaji Wakita, Akihide Ryo, Tetsuro Suzuki, Yoshiharu Matsuura, Nobuyuki Enomoto & Kohji Moriishi

    Scientific Reports 5, Article number: 17047 (2015)
    doi:10.1038/srep17047
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        Hepatitis B | Hepatitis B virus

Received:
    30 July 2015
Accepted:
    07 October 2015
Published online:
    23 November 2015

Abstract

Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.
Introduction

Hepatitis B virus (HBV) chronically infects approximately 3.4% of the world’s population and is a major factor for hepatocellular carcinoma following liver cirrhosis1. Interferon-alpha or nucleot(s)ide analogue inhibitors against the viral reverse transcriptase are currently approved for therapy for hepatitis B patients; however, these therapies are not necessarily effective for all such patients due to side effects and the emergence of escape mutant virus2. Thus, the development of new antiviral drugs that target several factors is still needed to prevent the liver diseases caused by HBV infection. Reliable and inexpensive cell culture systems and animal models of HBV infection are required in investigations of the underlying infection mechanism and pathogenesis of HBV. Although primary human hepatocytes (PHH), primary Tupaia belangeri hepatocytes (PTH), and the HepaRG cell line3 have been used as ex vivo HBV infection systems, these are typically employed under limited conditions, are expensive, and have difficulties maintaining stable susceptibility to HBV infection.

The HBV nucleocapsid is enveloped by a lipid bilayer enclosed within glycoproteins: the large (L), middle (M), and small (S) proteins of the HBV surface antigen (HBs)4. The L protein consists of preS1 and preS2 domains and the S protein, while the M protein consists of the preS2 domain and the S protein4. The S protein of the HBV virion has been shown initially, but weakly, to attach to heparan sulfate proteoglycans on hepatocytes5,6. Infection by HBV or hepatitis D virus (HDV) was previously reported to be neutralized by the antibody reacting to the preS1 region7 or by the myristoylated or acylated synthetic peptide composed of 47 N-terminal amino acids of the preS1 domain8,9,10, suggesting that the preS1 domain of the L protein is responsible for binding to the putative entry receptor(s). The sodium taurocholate cotransporting polypeptide (NTCP) was recently identified as a functional receptor for HBV and HDV because the myristoylated N-terminal region of the preS1 domain bound to NTCP and expression of NTCP rendered the HepG2 cell line susceptible to HBV infection11. The N-terminally myristoylated synthetic peptide corresponding to the region spanning from amino acid residue (aa) 2 to 48 of preS1 has been shown to interact with NTCP with high affinity11. The region spanning from aa 157 to 165 of NTCP was responsible for HBV infection and preS1 binding, while the region from aa 84 to 87 was for HBV infection but not preS1 binding11,12,13,14, suggesting that the region from aa 84 to 87 plays a role in a post-attachment step. Differences in these regions may determine host specificity for a member of the family Hepadnaviridae. Previous studies also suggested that the expression of NTCP provides HBV infectivity in the HepG2 cell line11,15,16,17. In the reported models, HBV could infect NTCP-expressing hepatoma cell lines under adherent monolayer-cell conditions11,15,16,17. However, NTCP-expressing HepG2 cells showed susceptibility to HBV infection compared with the parent cell line HepG2, but its infectivity was not high, which was indicated in the review process11. Schulze et al. reported that treatment with EGTA increased HBV infectivity in HepaRG cells18, suggesting that loosening of cell-cell junctions may promote HBV infectivity. Several reports suggest that NTCP is mainly expressed at the basolateral membrane of hepatocytes19,20,21. Thus, we hypothesized that the sufficient disruption of cell-cell junctions would expose NTCP to HBV virions in the medium, thereby promoting infectivity.

In the present study, we found lateral expression of NTCP in HepG2 cells transfected with the human SLC10A1 gene (NTCP gene), investigated the effect of non-adherent cell conditions on HBV infection, and then established a novel cell culture system for NTCP-dependent HBV infection. We also examined the effects of several compounds on HBV infectivity by using our culture system.

StephenW

乙型肝炎病毒感染高效地经由人类牛磺胆酸钠cotransporting多肽非粘附肝癌细胞

    奥山香织，土桥，弘毅开赛，智久田中，Atsuya山下，俊安本，文家陈彻冈本，前川真也，幸一渡，鹰二胁田，昭英良，铃木哲郎，松浦善治，榎本伸行和Kohji Moriishi

    科学报告5，商品编号：17047（2015年）
    DOI：10.1038 / srep17047
    下载文献
        乙肝|乙肝病毒

收稿日期：
    2015年7月30日
公认：
    2015年10月07日
网络发布时间：
    2015年11月23日

抽象

牛磺胆酸钠cotransporting多肽（NTCP）已被报告为功能性受体对乙型肝炎病毒（HBV）感染。然而，乙肝病毒无法有效感染HepG2细胞单层贴壁细胞的条件下表达NTCP（NTCP-HepG2细胞）。在这项研究中，NTCP主要定位在基底外侧膜区域，但不是根尖站点单层NTCP-HepG2细胞，。我们推测感染的非粘附细胞的条件将加强的HBV感染性。非粘附NTCP-HepG2细胞通过用胰蛋白酶和EDTA，其不降解NTCP在膜部分治疗制备。 HBV感染成功的NTCP-HepG2细胞在病毒剂量下在非粘附相中比在粘附期的10倍。非粘附NTCP-HepG2细胞与有效感染血源性或细胞培养衍生的HBV观察，并显着损害的肉豆蔻酰化前S1肽的存在。乙肝病毒也能有效感染非粘附细胞的条件下HepaRG细胞。我们筛选用我们的培养系统，并确定proscillaridin A作为有效的抗乙型肝炎病毒剂为7.2纳米的IC 50值的几种化合物。总之，感染的非粘附宿主细胞的条件增加的HBV感染在NTCP依赖性，因此提供了一种新的策略，以确定抗HBV药物和研究HBV感染的机制。
介绍

乙型肝炎病毒（HBV）慢性感染世界人口的约3.4％，并且是肝cirrhosis1肝细胞癌的主要因素。干扰素-α或nucleot（多个）对病毒逆转录酶的ide类似物抑制剂目前被批准用于治疗B型肝炎的患者;然而，这些疗法都未必有效由于副作用和逃逸突变virus2出现所有此类患者。因此，新的抗病毒药物的开发为目标的几个因素仍然需要防止由HBV感染的肝脏疾病。可靠和廉价的细胞培养系统和HBV感染的动物模型中需要的HBV的潜在感染机制和发病机理的研究。虽然原代人肝细胞（PHH），原发性中缅鼩肝细胞（PTH），而HepaRG细胞line3中已被用作离体HBV感染的系统，这些通常是有限的条件下使用的，是昂贵的，并有困难维持稳定的易感性的HBV感染。

的大（L），中（M），并且所述HBV表面抗原（HBs）中和4小（S）的蛋白质：乙肝病毒核衣壳由糖蛋白内封入脂质双层包围。 L蛋白是由前S1和前S2域和S蛋白，而M蛋白包含preS2区域和S protein4的。乙肝病毒的S蛋白已经初步显示出，但弱，附着于硫酸乙酰肝素蛋白多糖的hepatocytes5,6。感染HBV或肝炎病毒（HDV）的先前报道通过抗体反应前S1 region7或由肉豆蔻酰化或酰化合成肽的前S1 domain8,9,10 47 N-末端氨基酸组成的被中和，提示该L蛋白的前S1结构域负责结合推定的条目受体（多个）。的牛磺胆酸钠cotransporting多肽（NTCP）最近被鉴定为功能性受体的HBV和HDV因为前S1域绑定NTCP和NTCP的表达呈现的HepG2细胞易感于HBV infection11的肉豆蔻酰化的N-末端区域。对应于区域从氨基酸残基（AA）跨越2〜48前S1的N-末端肉豆蔻酰化合成肽已被证明与NTCP交互以高affinity11。该区域从氨基酸157跨越到NTCP 165负责HBV感染和前S1结合，而从氨基酸84至87的区域是HBV感染，但不前S1 binding11,12,13,14，这表明该区域从氨基酸84至87起着在后附接步骤的作用。在这些区域中的差异可确定宿主特异性为家庭嗜肝中的一员。以往的研究也表明，NTCP的表达提供了HBV感染的肝癌细胞line11,15,16,17。在报告的模型，乙肝病毒能够感染NTCP表达下贴壁单层细胞conditions11,15,16,17肝癌细胞系。然而，NTCP表达HepG2细胞显示敏感性HBV感染的亲代细胞系HepG2相比，但它的感染性不高，这是显示在审查process11。舒尔茨等。报告说，用EGTA治疗增加的HBV感染在HepaRG cells18，表明松动细胞 - 细胞连接的可促进HBV感染性。一些报告表明，NTCP主要表现在hepatocytes19,20,21的基底膜。因此，我们假设，细胞 - 细胞连接的破坏足以将暴露NTCP对HBV病毒粒子在介质中，从而促进感染性。

在目前的研究中，我们发现NTCP的转染人SLC10A1基因（NTCP的基因）HepG2细胞横向表达，调查了对HBV感染非粘附细胞条件的影响，然后再建立一个新的细胞培养体系NTCP依赖性HBV感染。我们还通过使用我们的培养系统中检验对HBV感染的几种化合物的效果。

StephenW

全文:http://www.nature.com/articles/srep17047