Squamous cell carcinoma antigen 1-mediated binding of hepatitis B virus to hepatocytes does not involve the hepatic serpin clearance system

The cellular receptor for hepatitis B virus (HBV) has not yet been identified. A recent candidate is a homologue of squamous cell carcinoma antigen 1 (SCCA1), a serpin. This study confirms that transfection of SCCA1 into mammalian cells (both hepatocyte-derived and of non-hepatocyte origin) results in increased HBV binding. Furthermore, virus bound to transfected cells is protected significantly from degradation by trypsin (75% compared with 30% in untransfected cells). The possibility that HBV enters cells via the hepatic clearance system for serpin-enzyme complexes was investigated by analysis of the reactive site loop of SCCA1. Functional and deletion mutants of SCCA1 were constructed by site-directed mutagenesis and compared with the wild type construct. In no case was virus binding reduced by functional alterations or deletions within the reactive site loop. A possible role for the low density lipoprotein receptor-related protein (LRP) in binding virus was investigated. SCCA1 transfection of Huh7 cells was shown to result in up-regulation of LRP expression, reaching levels observed in total liver. However, the use of receptor-associated protein (RAP), a competitive ligand for LRP, suggests than LRP up-regulation is not responsible for enhanced virus binding to SCCA1-transfected cells.     

Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells

A direct involvement of the hepatitis B virus (HBV) preS1-(21-47) sequence in virus attachment to cell membrane receptor(s) and the presence on the plasma membranes of HepG2 cells of protein(s) with receptor activity for HBV have been suggested by many previous experiments. In this study, by using a tetravalent derivative of the preS1-(21-47) sequence, we have isolated by affinity chromatography from detergent-solubilized HepG2 plasma membranes a 44-kDa protein (HBV-binding protein; HBV-BP), which was found to closely correspond to the human squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease inhibitors. Comparison of SCCA1 sequence with the sequence of the corresponding HBV-BP cDNA, cloned by polymerase chain reaction starting from RNA poly(A)(+) fractions extracted from HepG2 cells, indicated the presence of only four nucleotide substitutions in the coding region, leading to three amino acid changes. Intact recombinant HBV-BP lacked inhibitory activity for serine proteases such as alpha-chymotrypsin and trypsin but inhibited with high potency cysteine proteases such as papain and cathepsin L. Direct binding experiments confirmed the interaction of recombinant HBV-BP with the HBV preS1 domain. HepG2 cells overexpressing HBV-BP after transfection of corresponding cDNA showed a virus binding capacity increased by 2 orders of magnitude compared with untransfected cells, while Chinese hamster ovary cells, which normally do not bind to HBV, acquired susceptibility to HBV binding after transfection. Native HBV particle entry was enhanced in transfected cells. Both recombinant HBV-BP and antibodies to recombinant HBV-BP blocked virus binding and internalization in transfected cells as well as in primary human hepatocytes in a dose-dependent manner. Our findings suggest that this protein plays a major role in HBV infection.     

Expression of squamous cell carcinoma antigen-1 in liver enhances the uptake of hepatitis B virus envelope-derived bio-nanocapsules in transgenic rats

We previously developed the bio-nanocapsule, which consists of hepatitis B virus envelope L proteins. The bio-nanocapsule can be used to deliver genes and drugs specifically to the human liver-derived tissues in xenograft models, presumably by utilizing the human liver-specific mechanism of hepatitis B virus infection. The hepatitis B virus tropism is highly restricted to humans and higher primates. Thus, to evaluate the in vivo therapeutic effects of forthcoming bio-nanocapsule-based medicines, it will be crucial to develop an animal model whose liver is susceptible to both bio-nanocapsule and hepatitis B virus. In the present study, we aimed to establish a bio-nanocapsule-susceptible animal model using transgenic rats expressing squamous cell carcinoma antigen-1 (SCCA1), which has been proposed to be a receptor for hepatitis B virus, interacting with the hepatitis B virus envelope protein and enhancing the cellular uptake of hepatitis B virus. We show that the recombinant SCCA1 protein interacts directly with bio-nanocapsule and inhibits its attachment to the cultured human liver-derived cells. Furthermore, we have established a transgenic rat that specifically expresses SCCA1 in the liver and also demonstrate that the amount of bio-nanocapsule accumulated in the liver is significantly increased by the SCCA1 expression. Histological analysis suggests that bio-nanocapsule is preferentially incorporated into the SCCA1-expressing hepatocytes but not into macrophages, such as Küppfer cells, nor into endothelial cells. Therefore, this animal model is expected to be useful for the development of bio-nanocapsule-based medicines.     

Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis

The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.     

Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are major sources of acute and chronic hepatitis. HDV requires the envelope proteins of HBV for the processes of assembly and infection of new cells. Both viruses are able to infect hepatocytes though previous studies have failed to determine the mechanism of entry into such cells. This study began with evidence that suramin, a symmetrical hexasulfated napthylurea, could block HDV entry into primary human hepatocytes (PHH) and was then extrapolated to incorporate findings of others that suramin is one of many compounds that can block activation of purinergic receptors. Thus other inhibitors, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) and brilliant blue G (BBG), both structurally unrelated to suramin, were tested and found to inhibit HDV and HBV infections of PHH. BBG, unlike suramin and PPADS, is known to be more specific for just one purinergic receptor, P2X7. These studies provide the first evidence that purinergic receptor functionality is necessary for virus entry. Furthermore, since P2X7 activation is known to be a major component of inflammatory responses, it is proposed that HDV and HBV attachment to susceptible cells, might also contribute to inflammation in the liver, that is, hepatitis.     

Role of glycosaminoglycans for binding and infection of hepatitis B virus

Many parts of the life cycle of hepatitis B virus (HBV) infection of hepatocytes have been unravelled, but the attachment and entry process leading to infection is largely unknown. Using primary Tupaia hepatocyte cultures as an in vitro infection system, we determined that HBV uses cell-surface heparan sulfate proteoglycans as low-affinity receptor, because HBV infection was inhibited by heparin (IC50: 5 μg ml⁻¹) or other higher-sulfated polymers, but not by lower-sulfated glycosaminoglycans, such as chondroitin sulfate. Pretreatment of primary hepatocytes with heparinase decreased viral binding and inhibited HBV infection completely. Interestingly, after preS1-dependent viral binding at 16°C to the cell surface, subsequent infection could still be inhibited by HBV preS1-lipopeptides, but not by heparin any more, suggesting a shift of the virus to a high-affinity receptor. In summary, we suggest following multistep attachment process: in vivo , HBV is initially trapped within the liver in the space of Dissé by heparan sulfate proteoglycans. Thereafter, HBV binds via its preS1 attachment site and the N-terminal myristic acid to a yet unknown, high-affinity receptor that confers uptake in a yet unknown compartment.     

Inhibition of hepatitis B virus in mice by RNA interference

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.     

Inhibitory effect of silibinin on hepatitis B virus entry

Hepatitis B virus (HBV) infection is a worldwide health problem because of its potential to cause liver cirrhosis and hepatocellular carcinoma. Silibinin is a constituent of an extract of milk thistle, which is empirically used as a herbal medicine for the protection of liver, but its detailed effects on HBV are unknown. Because a previous study reported that silibinin hinders clathlin-mediated endocytosis (CME), we aimed to test whether silibinin inhibits the entry of HBV into hepatocytes. Using HepG2-NTCP-C4 cells, which overexpress sodium taurocholate cotransporting polypeptide (NTCP), it was shown that silibinin inhibited HBV infection dose-dependently. Similar effects were observed using human primary hepatocytes (PXB-cells). Additionally, a combination of silibinin and entecavir reduced HBV DNA in the culture supernatant more than either mono-treatment alone in HepG2-NTCP-C4 cells that had already been infected with HBV. Silibinin decreased transferrin uptake but did not affect the interaction between the HBV envelope and NTCP, suggesting that silibinin might inhibit HBV infection by hindering CME. In conclusion, this study showed that silibinin inhibits HBV entry in vitro.     

Enhanced inhibition of hepatitis B virus production by asialoglycoprotein receptor-directed interferon

Most chronic carriers of hepatitis B virus (HBV) do not respond to interferon (IFN) treatment. This limitation of IFN therapy may be due in part to scant expression of IFN receptor in the liver. Because the asialoglycoprotein (ASGP) receptor is specifically expressed in the liver at high density, the ASGP receptor-binding domain was generated within an N-glycosylated human IFN-beta molecule by the removal of sialic acid to direct this cytokine to the liver. This modified IFN (asialo-IFN-beta) demonstrated greater inhibition of HBV production in ASGP receptor-positive human liver cells transfected with a replication-competent HBV construct than did conventional IFN-alpha or IFN-beta. Furthermore, the enhanced antiviral effect of asialo-IFN-beta was supported by induction of the 2'-5' oligoadenylate synthetase, an indicator of IFN activity, at a level significantly higher than that produced by conventional IFN-beta. Moreover, mouse asialo-IFN-beta profoundly reduced viremia in vivo in HBV-transfected athymic nude mice, in contrast to conventional IFN-beta, which had no substantial effect. These experiments demonstrate that directing IFN to ASGP receptor facilitates its signaling in the liver and augments its antiviral effect, and is therefore useful in overcoming the limited antiviral effect of conventional IFNs.     

Overcoming tolerance in hepatitis B virus transgenic mice: a possible involvement of regulatory T cells

The hepatitis B virus (HBV) transgenic mouse (Tg) 50-4 strain is immunologically tolerant to HBV antigens. Various vaccination strategies have been attempted but failed to break the tolerance in the mouse. Although the tolerance to HBV antigen is maintained, this mouse strain develops spontaneous liver disease beginning at the age of about 3 months. We attempted to induce immune responses to HBV surface antigen (HBsAg) in the Tg by immunization with recombinant vaccinia virus expressing HBsAg (vvHBV), and observed different immunological responsiveness between 2-month-old and 5-month-old Tg. In contrast to the unbreakable tolerance reported previously, we could induce both the cytotoxic T lymphocyte (CTL) and the antibody response against HBsAg by the vvHBV immunization. The cytokine expression pattern indicated that T helper 1 type immune response was induced. However, interestingly, these immune responses were observed only in the 5-month-old Tg, but not in the 2-month-old Tg. Furthermore, CD4+ T cells from 2-month-old mice, but not those from 5-month-old mice, inhibited CTL response to HBV antigen when adoptively transferred to C57BL/6. These results suggest the possible involvement of regulatory T cell function in the HBV Tg for maintaining tolerance. This study would contribute to a better understanding of immune status of the HBV Tg as a model of human chronic hepatitis and to the search for new therapeutic targets for chronic viral infections.     

Direct injection of hepatitis B virus DNA into liver induced hepatitis in adult rats

Hepatitis B virus (HBV) surface antigen (HBsAg) genes were injected directly into the liver of adult rats with non-histone chromosomal protein high mobility group 1 by the hemagglutinating B virus of Japan (Sendai virus)-liposome method (Kato, K., Nakanishi, M., Kaneda, Y., Uchida, T., and Okada, Y. (1991) J. Biol. Chem. 266, 3361-3364). Immunohistochemical analysis showed that HBV surface antigen was expressed by the hepatocytes in vivo. On successive injections of the HBsAg genes, the antibody to HBV surface polypeptides was produced in the rats, and characteristic pathological changes of lymphocytic focal necrosis and denaturation of hepatic cells were observed in the liver of all the rats. We conclude that hepatitis is caused by the direct injection of HBsAg genes.     

Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus DNA

Persistent infection by hepatitis B virus (HBV) is epidemiologically correlated with the prevalence of hepatocellular carcinoma, but its role in tumor development is not yet understood. To study the putative oncogenic potential of HBV, a non-malignant immortal mouse hepatocyte line FMH202 harboring metallothionein promoter-driven simian virus 40 large tumor antigen was transfected with HBV DNA. All stably transfected clones which replicated HBV displayed malignant growth characteristics in soft agar and were tumorigenic upon inoculation in nude mice. The nude mice tumors were histologically classified as differentiated or anaplastic hepatocellular carcinomas. As with human liver carcinomas, rearrangements of in vitro integrated HBV sequences were observed in the nude mouse tumors, and in tumor-derived cell lines. In one case, expression of viral core and surface antigens was blocked in the tumors, correlating with hypermethylation of the HBV genome. However, the expression of X gene was maintained in most tumors and tumor-derived cell lines. X protein was detected in nuclei by immune fluorescence and by immune blot. These results provide the first demonstration that HBV displays oncogenic potential in an experimental system. This system could be useful to functionally identify HBV genes which convey a tumorigenic phenotype.     

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Molecular Determinants of Hepatitis B and D Virus Entry Restriction in Mouse Sodium Taurocholate Cotransporting Polypeptide

Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), primarily infect humans, chimpanzees, or tree shrews (Tupaia belangeri). Viral infections in other species are known to be mainly restricted at the entry level since viral replication can be achieved in the cells by transfection of the viral genome. Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV and HDV, and amino acids 157 to 165 of NTCP are critical for viral entry and likely limit viral infection of macaques. However, the molecular determinants for viral entry restriction in mouse NTCP (mNTCP) remain unclear. In this study, mNTCP was found to be unable to support either HBV or HDV infection, although it can bind to pre-S1 of HBV L protein and is functional in transporting substrate taurocholate; comprehensive swapping and point mutations of human NTCP (hNTCP) and mNTCP revealed molecular determinants restricting mNTCP for viral entry of HBV and HDV. Remarkably, when mNTCP residues 84 to 87 were substituted by human counterparts, mNTCP can effectively support viral infections. In addition, a number of cell lines, regardless of their species or tissue origin, supported HDV infection when transfected with hNTCP or mNTCP with residues 84 to 87 replaced by human counterparts, highlighting the central role of NTCP for viral infections mediated by HBV envelope proteins. These studies advance our understanding of NTCP-mediated viral entry of HBV and HDV and have important implications for developing the mouse model for their infections.