Florian A. Lempp

Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.

BACKGROUND & AIMS Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type-related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor

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 8 μM. 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.

Impaired uptake of conjugated bile acids and hepatitis b virus pres1‐binding in na+‐taurocholate cotransporting polypeptide knockout mice

The Na+‐taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte‐specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1‐knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1−/− hepatocytes showed absence of sodium‐dependent taurocholic acid uptake, whereas sodium‐independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1−/− mice, explained by increased Asbt and Ostα/β expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down‐regulation of OATP1A1 and up‐regulation of OATP1A4. Furthermore, sodium‐dependent taurocholic acid uptake was inhibited by Myrcludex B in wild‐type hepatocytes, while Slc10a1−/− hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1‐/‐ mice. Conclusion: The Slc10a1‐knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP‐independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation. (Hepatology 2015;62:207–219)

Inhibitors of Hepatitis B Virus Attachment and Entry

Inhibition of virus entry has become a major concept in the development of new antiviral drugs. Entry inhibitors can either neutralize activities of viral surface proteins or target essential host factors such as (co)receptors. Due to its distinct tissue tropism and the highly specific viral and cellular factors involved in its entry, hepatitis B virus (HBV) is an ideal candidate for entry inhibition. Hepatitis B immunoglobulins neutralize infection by binding to the S-domain of HBV surface proteins and are used to prevent reinfection of the graft after liver transplantation. Novel S or preS-specific monoclonal antibodies are currently in development. The identification of sodium-taurocholate cotransporting polypeptide (NTCP) as a bona fide receptor has revealed a suitable target for HBV entry inhibition. NTCP receptor function is blocked by a variety of different agents including Myrcludex B, a synthetic N-acylated preS1-derived lipopeptide that inhibits HBV entry in vitro and in vivo with high efficacy. Current antiviral treatment for chronic HBV-infected patients focuses on the inhibition of the viral polymerase via nucleos(t)ide analogues (NA). Entry inhibitors in combination with NAs could block reinfection and shield naive hepatocytes that emerge from natural liver turnover, opening up new therapeutic options.

Hepatitis delta virus: insights into a peculiar pathogen and novel treatment options

Chronic hepatitis D is the most severe form of viral hepatitis, affecting ∼20 million HBV-infected people worldwide. The causative agent, hepatitis delta virus (HDV), is a unique human pathogen: it is the smallest known virus; it depends on HBV to disseminate its viroid-like RNA; it encodes only one protein (HDAg), which has both structural and regulatory functions; and it replicates using predominantly host proteins. The failure of HBV-specific nucleoside analogues to suppress the HBV helper function, and the limitations of experimental systems to study the HDV life cycle, have impeded the development of HDV-specific drugs. Thus, the only clinical regimen for HDV is IFNα, which shows some efficacy but long-term virological responses are rare. Insights into the receptor-mediated entry of HDV, and the observation that HDV assembly requires farnesyltransferase, have enabled novel therapeutic strategies to be developed. Interference with entry, for example through blockade of the HBV–HDV-specific receptor sodium/taurocholate cotransporting polypeptide NTCP by Myrcludex B, and inhibition of assembly by blockade of farnesyltransferase using lonafarnib or nucleic acid polymers such as REP 2139-Ca, have shown promising results in phase II studies. In this Review, we summarize our knowledge of HDV epidemiology, pathogenesis and molecular biology, with a particular emphasis on possible future developments.

Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor

BACKGROUND & AIMS Hepatitis B virus (HBV) is a major human pathogen restricted to hepatocytes. Expression of the specific receptor human sodium taurocholate cotransporting polypeptide (hNTCP) in mouse hepatocytes renders them susceptible to hepatitis delta virus (HDV), a satellite of HBV; however, HBV remains restricted at an early stage of replication. This study aims at clarifying whether this restriction is caused by the lack of a dependency factor or the activity of a restriction factor. METHODS Six hNTCP-expressing mouse and human cell lines were generated and functionally characterized. By fusion with replication-supporting but non-infectable HepG2 cells, we analysed the ability of these heterokaryonic cells to fully support HBV replication by HBcAg expression and HBsAg/HBeAg secretion. RESULTS While hNTCP expression in three mouse cell lines and the non-hepatic human HeLa cells conferred susceptibility to HDV, HBV replication was still restricted. Upon fusion of refractive cells to HepG2 cells, all heterokaryonic cells supported receptor-mediated infection with HBV. hNTCP was provided by the mouse cells and replication competence came from the HepG2 cell line. Transfection of a covalently closed circular DNA (cccDNA)-like molecule into non-susceptible cells promoted gene expression, indicating that the limiting step is upstream of cccDNA formation. CONCLUSIONS In addition to the expression of hNTCP, establishment of HBV infection in mouse and non-hepatocytic human cell lines requires supplementation with a dependency factor and is not limited by a restriction factor. This result opens new avenues for the development of a fully permissive immunocompetent HBV mouse model.

Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes

Infections with the human hepatitis B virus (HBV) and hepatitis D virus (HDV) depend on species‐specific host factors like the receptor human sodium taurocholate cotransporting polypeptide (hNTCP). Complementation of mouse hepatocytes with hNTCP confers susceptibility to HDV but not HBV, indicating the requirement of additional HBV‐specific factors. As an essential premise toward the establishment of an HBV‐susceptible animal model, we investigated the role of hNTCP as a limiting factor of hepatocytes in commonly used laboratory animals. Primary hepatocytes from mice, rats, dogs, pigs, rhesus macaques, and cynomolgus macaques were transduced with adeno‐associated viral vectors encoding hNTCP and subsequently infected with HBV. Cells were analyzed for Myrcludex B binding, taurocholate uptake, HBV covalently closed circular DNA formation, and expression of all HBV markers. Sodium taurocholate cotransporting polypeptide (Ntcp) from the respective species was cloned and analyzed for HBV and HDV receptor activity in a permissive hepatoma cell line. Expression of hNTCP in mouse, rat, and dog hepatocytes permits HDV infection but does not allow establishment of HBV infection. Contrarily, hepatocytes from cynomolgus macaques, rhesus macaques, and pigs became fully susceptible to HBV upon hNTCP expression with efficiencies comparable to human hepatocytes. Analysis of cloned Ntcp from all species revealed a pronounced role of the human homologue to support HBV and HDV infection. Conclusion: Ntcp is the key host factor limiting HBV infection in cynomolgus and rhesus macaques and in pigs. In rodents (mouse, rat) and dogs, transfer of hNTCP supports viral entry but additional host factors are required for the establishment of HBV infection. This finding paves the way for the development of macaques and pigs as immunocompetent animal models to study HBV infection in vivo, immunological responses against the virus and viral pathogenesis. (Hepatology 2017;66:703–716).

Hepatitis B Virus Infection of a Mouse Hepatic Cell Line Reconstituted with Human Sodium Taurocholate Cotransporting Polypeptide

ABSTRACT Hepatitis B virus (HBV) enters hepatocytes via its receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). So far, HBV infection has been achieved only in human hepatic cells reconstituted with hNTCP and not in cells of mouse origin. Here, the first mouse liver cell line (AML12) which gains susceptibility to HBV upon hNTCP expression is described. Thus, HBV infection of receptor-expressing mouse hepatocytes does not principally require a human cofactor but can be triggered by endogenous murine determinants.

Hepatitis Delta Virus: Replication Strategy and Upcoming Therapeutic Options for a Neglected Human Pathogen

The human Hepatitis Delta Virus (HDV) is unique among all viral pathogens. Encoding only one protein (Hepatitis Delta Antigen; HDAg) within its viroid-like self-complementary RNA, HDV constitutes the smallest known virus in the animal kingdom. To disseminate in its host, HDV depends on a helper virus, the human Hepatitis B virus (HBV), which provides the envelope proteins required for HDV assembly. HDV affects an estimated 15–20 million out of the 240 million chronic HBV-carriers and disperses unequally in disparate geographical regions of the world. The disease it causes (chronic Hepatitis D) presents as the most severe form of viral hepatitis, leading to accelerated progression of liver dysfunction including cirrhosis and hepatocellular carcinoma and a high mortality rate. The lack of approved drugs interfering with specific steps of HDV replication poses a high burden for gaining insights into the molecular biology of the virus and, consequently, the development of specific novel medications that resiliently control HDV replication or, in the best case, functionally cure HDV infection or HBV/HDV co-infection. This review summarizes our current knowledge of HBV molecular biology, presents an update on novel cell culture and animal models to study the virus and provides updates on the clinical development of the three developmental drugs Lonafarnib, REP2139-Ca and Myrcludex B.