NTCP and Beyond: Opening the Door to Unveil Hepatitis B Virus Entry
通过亲和纯化和质谱分析，以HBV pres1衍生的脂肽作为诱饵，他们确定Tupaia belangeri NTCP (tsNTCP)是与脂肽相互作用的细胞因子。NTCP是一种位于肝细胞基底外侧膜上的转运蛋白，主要参与肝摄取结合的胆汁酸盐(见下文)。脂肽被证实能特异性结合人类NTCP (hNTCP)和tsNTCP，但令人惊讶的是，与HBV感染的物种特异性相关的非食蟹猴NTCP (mkNTCP)却不具有特异性:HBV能够有效地感染人类和Tupaia，而不具有食蟹猴。
有趣的是，这一结果也与该肽与各自原代肝细胞及其体内向肝性的体外结合活性相关。NTCP在HBV、其卫星病毒HDV以及与HBV关系密切的灵长类动物hepadnavirus wooly monkey HBV病毒感染中的作用通过敲除和过表达分析进一步探讨[9,38,39]。sirna介导的NTCP在原发性人肝细胞(PHH)、原发性Tupaia肝细胞和分化肝细胞中的敲除降低了HBV和HDV感染，而异位表达NTCP在HepG2细胞中赋予了HBV敏感性，这原本并不支持高效的感染。这有力地证明了NTCP是HBV感染的一个重要因素。
Sodium Taurocholate Co-Transporting Polypeptide (NTCP) as a Bona Fide HBV Receptor One of the recent milestones in the field in HBV molecular biology is the identification of NTCP as a host entry receptor, as reported by Yan and Zhong et al. in late 2012 . By affinity purification and mass spectrometry analysis using an HBV preS1-derived lipopeptide as bait, they identified Tupaia belangeri NTCP (tsNTCP) as a cellular factor interacting with this lipopeptide. NTCP is a transporter residing in the basolateral membrane of hepatocytes and is involved in the hepatic uptake of mostly conjugated bile salts (see below). The lipopeptide was confirmed to specifically bind human NTCP (hNTCP), as well as tsNTCP, but surprisingly not crab-eating monkey NTCP (mkNTCP), which correlated with the species specificity of HBV infection: HBV is able to efficiently infect humans and Tupaia, but not crab-eating monkey . Interestingly, this result also correlated with the in vitro binding activity of the peptide to the respective primary hepatocytes  and their in vivo hepatotropism . The role of NTCP in the viral infection of HBV, its satellite virus, HDV, and a closely related primate hepadnavirus wooly monkey HBV was further examined by knockdown and overexpression analyses [9,38,39]. siRNA-mediated knockdown of NTCP in primary human hepatocytes (PHH), primary Tupaia hepatocytes and differentiated HepaRG cells reduced HBV and HDV infection, while ectopic expression of NTCP conferred HBV susceptibility in HepG2 cells, which originally did not support efficient infection . This strongly argues that NTCP is an essential factor for HBV infection. The expression of NTCP in different cells was consistent with the HBV susceptibility, as it was significantly expressed in HBV-susceptible cells, PHH and differentiated HepaRG cells, but was weakly expressed or absent in HepG2, Huh-7, FLC4 and HeLa cells, which show little to no infection [40–42]. The introduction of NTCP into Huh-7 and undifferentiated HepaRG cells conferred HBV infection to these cells to some extent . Although the total expressions in these transduced cells were comparable, hNTCP-expressing HepG2 cells showed much higher infection efficiency when compared with other human hepatocyte cell lines [38,43,44]. In the initial study, infection efficiency was ~10% in NTCP-overexpressing HepG2 cells cultured with medium containing 2% dimethyl sulfoxide (DMSO) . Subsequent analysis showed that increasing the DMSO concentration to more than 2.5%~3% augmented infection efficiency to 50%~70%, as evaluated by immunofluorescence of HBV proteins, although the virus inoculum was different in these studies [38,43]. The speculations include that DMSO augmented the gene expression of NTCP, promoted the membrane localization of NTCP and changed the post-translational modification of NTCP, but the detailed molecular mechanisms for DMSO-mediated promotion of HBV infection is open for further studies. It remains unknown why not all of the cells were infected with HBV in these reports, but it is possible that the NTCP function for supporting HBV entry is reflected by post-translational modification, subcellular localization or other factors that are governed by cell conditions or by more general conditions, such as the cell cycle, cellular microenvironment or architecture. Another open question is on the high susceptibility for HDV, but not HBV, in Huh-7 cells overexpressing hNTCP [9,38]. Future analysis of this issue is necessary in order to establish a cell culture model that is 100% susceptible to HBV infection.
compounds known to be NTCP inhibitors, including progesterone, propranolol and bosentan, have been shown to block HBV infection (Figure 1) . NTCP substrates, such as taurocholate, tauroursodeoxycholate and bromosulfophthalein, also inhibited HBV infection [38,42,68]. An anticholesteremic drug, ezetimibe, has been shown to block HBV entry , and this drug was reported to inhibit the NTCP transporter . These results indicate that compounds modulating NTCP function could substantially inhibit HBV infection. HepG2 cells engineered to overexpress NTCP are also useful for high-throughput screening to identify compounds targeting NTCP and inhibiting HBV infection. One example identified in such chemical screening is the oxysterols, which are oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis .
Host-targeting antivirals are generally expected to have significant advantages, including a much lower frequency drug resistance, universal antiviral effects beyond viral genotypes and complementary mechanisms of action that might act in a synergistic manner with currently available antiviral agents . More importantly, they offer an additional therapeutic choice, given that only IFNs and nucleoside analogs are currently available as anti-HBV agents.
Identification of NTCP as an HBV entry receptor has accelerated the understanding of HBV molecular biology and offered useful experimental systems to analyze the HBV and HDV lifecycle, including the identification of host restriction and dependency factors. NTCP also represents a new therapeutic target in the development of new anti-HBV agents. Further analyses using a new cell culture system are necessary in order to clarify the molecular mechanisms underlying NTCP-mediated HBV infection and to establish an in vivo small animal model that fully supports HBV infection.
NTCP and Beyond: Opening the Door to Unveil Hepatitis B Virus Entry https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958888/