Evaluation of the SARS-CoV-2 RNA detection reagent LAMPdirect Genelyzer KIT using nasopharyngeal swab and saliva samples.

The LAMPdirect Genelyzer KIT allows for the detection of SARS-CoV-2 RNA in saliva samples with a loop-mediated isothermal amplification (LAMP) method and generates results within 20 min. It has been approved by the Pharmaceuticals and Medical Devices Agency in Japan. In this study, the performance of the LAMPdirect Genelyzer KIT was compared with that of the RT-qPCR reference method using 50 nasopharyngeal swabs and 100 saliva samples. In addition, we evaluated the applicability of an alternative reverse transcriptase and the effects of an inactivation buffer. The total agreement rates were 80.0 % and 82.0 % for nasopharyngeal and saliva samples, respectively. When considering samples at the detection limit (50 copies/reaction) that increases the chance of transmission between humans, the total agreement rates were 100% and 94.1% for nasopharyngeal and saliva samples, respectively. The LAMP method is simple, fast, and inexpensive, making it useful for small medical institutions or rural areas.

A versatile method to profile hepatitis B virus DNA integration.

BACKGROUND: HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome. METHODS AND RESULTS: Illumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR. CONCLUSIONS: RAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.

Selective inhibition of hepatitis B virus internalization by oxysterol derivatives.

Given that the current approved anti-hepatitis B virus (HBV) drugs suppress virus replication and improve hepatitis but cannot eliminate HBV from infected patients, new anti-HBV agents with different mode of action are urgently needed. In this study, we identified a semi-synthetic oxysterol, Oxy185, that can prevent HBV infection in a HepG2-based cell line and primary human hepatocytes. Mechanistically, Oxy185 inhibited the internalization of HBV into cells without affecting virus attachment or replication. We also found that Oxy185 interacted with an HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and inhibited the oligomerization of NTCP to reduce the efficiency of HBV internalization. Consistent with this mechanism, Oxy185 also inhibited the hepatitis D virus infection, which relies on NTCP-dependent internalization, but not hepatitis A virus infection, and displayed pan-genotypic anti-HBV activity. Following oral administration in mice, Oxy185 showed sustained accumulation in the livers of the mice, along with a favorable liver-to-plasma ratio. Thus, Oxy185 is expected to serve as a useful tool compound in proof-of-principle studies for HBV entry inhibitors with this novel mode of action.

Amentoflavone inhibits hepatitis B virus infection via the suppression of preS1 binding to host cells.

Hepatitis B virus (HBV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Current therapeutic drugs for chronic HBV infection use IFN and nucleos(t)ide analogs; however, their efficacy is limited. Thus, there is an urgent need to develop new antivirals for HBV therapy. In this study, we identified a plant-derived polyphenolic bioflavonoid, amentoflavone, as a new anti-HBV compound. Amentoflavone treatment dose-dependently inhibited HBV infection in HBV-susceptible cells with HepG2-hNTCP-C4 and primary human hepatocyte PXB-cells. A mode-of-action study showed that amentoflavone inhibits the viral entry step, but not the viral internalization and early replication processes. Attachment of HBV particles as well as HBV preS1 peptide to HepG2-hNTCP-C4 cells was inhibited by amentoflavone. The transporter assay revealed that amentoflavone partly inhibits uptake of sodium taurocholate cotransporting polypeptide (NTCP)-mediated bile acid. Furthermore, effect of various amentoflavone analogs on HBs and HBe production from HBV-infected HepG2-hNTCP-C4 cells was examined. Robustaflavone exhibited comparable anti-HBV activity to that of amentoflavone and an amentoflavone-7,4', 4‴-trimethyl ether derivative (sciadopitysin) with moderate anti-HBV activity. Cupressuflavone or the monomeric flavonoid apigenin did not exhibit the antiviral activity. Amentoflavone and its structurally related biflavonoids may provide a potential drug scaffold in the design of a new anti-HBV drug inhibitor targeting NTCP.

Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus.

Hepatitis B virus (HBV) infection is a major public health problem. The sodium taurocholate cotransporting polypeptide (NTCP) has been identified as an essential HBV receptor. Human hepatocytes are infected with HBV via binding between the preS1 region of the HBV large envelope protein and the NTCP. However, the role of preS2 in HBV entry is not well understood. In this study, we induced anti-preS2 serum in mice by DNA immunization, and showed that the resulting antiserum neutralized HBV infectivity. Competition assays using overlapping peptides suggested that the neutralizing epitope is located in the N-terminal region of preS2. In addition, monoclonal antibodies targeting the N-terminal region of preS2 neutralized HBV infectivity, indicating that these domains are critical epitopes for viral neutralization. These findings provide new insights into the HBV entry machinery while suggesting a novel modality for the prevention and treatment of HBV infection.

Activities of endogenous APOBEC3s and uracil-DNA-glycosylase affect the hypermutation frequency of hepatitis B virus cccDNA.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) plays a key role in the persistence of viral infection. We have previously shown that overexpression of an antiviral factor APOBEC3G (A3G) induces hypermutation in duck HBV (DHBV) cccDNA, whereas uracil-DNA-glycosylase (UNG) reduces these mutations. In this study, using cell-culture systems, we examined whether endogenous A3s and UNG affect HBV cccDNA mutation frequency. IFNγ stimulation induced a significant increase in endogenous A3G expression and cccDNA hypermutation. UNG inhibition enhanced the IFNγ-mediated hypermutation frequency. Transfection of reconstructed cccDNA revealed that this enhanced hypermutation caused a reduction in viral replication. These results suggest that the balance of endogenous A3s and UNG activities affects HBV cccDNA mutation and replication competency.

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

NTCP Oligomerization Occurs Downstream of the NTCP-EGFR Interaction during Hepatitis B Virus Internalization.

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and noncompetitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, coimmunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affects the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. IMPORTANCE Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus.

Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.

MCPIP1 reduces HBV-RNA by targeting its epsilon structure.

Hepatitis B virus (HBV) is the major causative factor of chronic viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. We previously demonstrated that a proinflammatory cytokine IL-1ß reduced the level of HBV RNA. However, the mechanism underlying IL-1ß-mediated viral RNA reduction remains incompletely understood. In this study, we report that immune regulator Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) can reduce HBV RNA in hepatocytes. MCPIP1 expression level was higher in the liver tissue of HBV-infected patients and mice. Overexpression of MCPIP1 decreased HBV RNA, whereas ablating MCPIP1 in vitro enhanced HBV production. The domains responsible for RNase activity or oligomerization, were required for MCPIP1-mediated viral RNA reduction. The epsilon structure of HBV RNA was important for its antiviral activity and cleaved by MCPIP1 in the cell-free system. Lastly, knocking out MCPIP1 attenuated the anti-HBV effect of IL-1ß, suggesting that MCPIP1 is required for IL-1ß-mediated HBV RNA reduction. Overall, these results suggest that MCPIP1 may be involved in the antiviral effect downstream of IL-1ß.

EBV-LMP1 induces APOBEC3s and mitochondrial DNA hypermutation in nasopharyngeal cancer.

An Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a principal oncogene that plays a pivotal role in EBV-associated malignant tumors including nasopharyngeal cancer (NPC). Recent genomic landscape studies revealed that NPC also contained many genomic mutations, suggesting the role of LMP1 as a driver gene for the induction of these genomic mutations. Nonetheless, its exact mechanism has not been investigated. In this study, we report that LMP1 alters the expression profile of APOBEC3s(A3s), host deaminases that introduce consecutive C-to-U mutations (hypermutation). In vitro, LMP1 induces APOBEC3B (A3B) and 3F(A3F), in a nasopharyngeal cell line, AdAH. Overexpression of LMP1, A3B, or A3F induces mtDNA hypermutation, which is also detectable from NPC specimens. Expression of LMP1 and A3B in NPC was correlated with neck metastasis. These results provide evidence as to which LMP1 induces A3s and mtDNA hypermutation, and how LMP1 facilitates metastasis is also discussed.

Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.

BACKGROUND: Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. METHODS: We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. RESULTS: We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 µM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. CONCLUSIONS: We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

Concept of Viral Inhibitors via NTCP.

Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an entry receptor for hepatitis B and D viruses (HBV and HDV) has not only promoted our understanding of the mechanism underlying the viral entry process, but also provided cell culture models supporting viral infection. These models have greatly facilitated cell-based chemical screening for the discovery of entry inhibitors, and mode of action studies using such inhibitors have shown the advantages of NTCP as a drug target. Furthermore, in vitro chemical screening by application of high-throughput affinity-based technologies that target NTCP has identified a variety of unique small molecules that interfere with viral entry. This review summarizes this hot topic in the development of HBV/HDV entry inhibitors, with special focus on the use of NTCP as a drug target.

[From the Establishment of Hepatitis B Virus Cell Culture Systems to Drug Discovery].

The development of antiviral agents enables the control of chronic infectious diseases caused by infection with herpesviruses, human immunodeficiency virus, and hepatitis C virus. In contrast, antiviral treatment against hepatitis B virus (HBV) infection remains a significant area for improvement. One of the main barriers hampering the progress of HBV research has been a lack of cell culture systems efficiently reproducing the viral proliferation process. Recently, cell line-based HBV infection systems have been developed which are useful to analyze the mechanisms of HBV replication and to screen for new anti-HBV agents. In this article, we summarize the establishment of such cell models and the identification of small molecules that inhibit the HBV entry process and discuss their future potential as a novel class of anti-HBV agents.

A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species Specificity.

Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary "arms race" between hepadnaviruses and their hosts. Here, we present evidence suggesting that the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (dN/dS ratio [ratio of nonsynonymous to synonymous evolutionary changes] of <1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7% of NTCP amino acid residues exhibit rapid evolution under positive selection (dN/dS ratio of >1). Notably, a substitution at amino acid (aa) 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested a close association of the aa 158 positive selection with the pressure by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to the host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this NTCP residue. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing adaptive mutation in host receptor.IMPORTANCE HBV and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary arms race between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidence supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species specificity.

De Novo Macrocyclic Peptide Inhibitors of Hepatitis B Virus Cellular Entry.

Hepatitis B virus (HBV) constitutes a significant public health burden, and currently available treatment options are not generally curative, necessitating the development of new therapeutics. Here we have applied random non-standard peptide integrated discovery (RaPID) screening to identify small macrocyclic peptide inhibitors of HBV entry that target the cell-surface receptor for HBV, sodium taurocholate cotransporting polypeptide (NTCP). In addition to their anti-HBV activity, these molecules also inhibit cellular entry by the related hepatitis D virus (HDV), and are active against diverse strains of HBV (including clinically relevant nucleos(t)ide analog-resistant and vaccine escaping strains). Importantly, these macrocyclic peptides, in contrast to other NTCP-binding HBV entry inhibitors, exhibited no inhibition of NTCP-mediated bile acid uptake, making them appealing candidates for therapeutic development.

Development of a cell-based assay to identify hepatitis B virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide.

Sodium taurocholate cotransporting polypeptide (NTCP) is a major entry receptor of hepatitis B virus (HBV) and one of the most attractive targets for anti-HBV drugs. We developed a cell-mediated drug screening method to monitor NTCP expression on the cell surface by generating a HepG2 cell line with tetracycline-inducible expression of NTCP and a monoclonal antibody that specifically detects cell-surface NTCP. Using this system, we screened a small molecule library for compounds that protected against HBV infection by targeting NTCP. We found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface NTCP with an IC50 of ~40 µM. We also found that glabridin could attenuate the inhibitory effect of taurocholate on type I interferon signaling by depleting the level of cell-surface NTCP. These results demonstrate that our screening system could be a powerful tool for discovering drugs targeting HBV entry.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

Chemical array system, a platform to identify novel hepatitis B virus entry inhibitors targeting sodium taurocholate cotransporting polypeptide.

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.

Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes.

Current anti-hepatitis B virus (HBV) agents, which include nucleos(t)ide analogs and interferons, can significantly suppress HBV infection. However, there are limitations in the therapeutic efficacy of these agents, indicating the need to develop anti-HBV agents with different modes of action. In this study, through a functional cell-based chemical screening, we found that a thiazolidinedione, troglitazone, inhibits HBV infection independently of the compound's ligand activity for peroxisome proliferator-activated receptor γ (PPARγ). Analog analysis suggested chemical moiety required for the anti-HBV activity and identified ciglitazone as an analog having higher anti-HBV potency. Whereas, most of the reported HBV entry inhibitors target viral attachment to the cell surface, troglitazone blocked a process subsequent to viral attachment, i.e., internalization of HBV preS1 and its receptor, sodium taurocholate cotransporting polypeptide (NTCP). We also found that NTCP was markedly oligomerized in the presence of HBV preS1, but such NTCP oligomerization was abrogated by treatment with troglitazone, but not with pioglitazone, correlating with inhibition activity to viral internalization. Also, competitive peptides that blocked NTCP oligomerization impeded viral internalization and infection. This work represents the first report identifying small molecules and peptides that specifically inhibit the internalization of HBV. This study is also significant in proposing a possible role for NTCP oligomerization in viral entry, which will shed a light on a new aspect of the cellular mechanisms regulating HBV infection.