Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide

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Sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for hepatitis D virus (HDV) and its helper hepatitis B virus (HBV).In cultured cell lines, HDV infection through mouse NTCP is restricted by residues 84 to 87 of the receptor. This study shows that mice with these three amino acids altered their corresponding human residues (H84R, T86K, and S87N) in endogenous mouse NTCP support de novo HDV infection in vivo. HDV infection was documented by the presence of replicative forms of HDV RNA and HDV proteins in liver cells at day 6 after viral inoculation. Monoclonal antibody specifically binding to the motif centered on K86 in NTCP partially inhibited HDV infection. These studies demonstrated specific interaction between the receptor and the viral envelopes in vivo and established a novel mouse model with minimal genetic manipulation for studying HDV infection. The model will also be useful for evaluating entry inhibitors against HDV and its helper HBV. Sodium taurocholate cotransporting polypeptide (NTCP), a bile acids transporter in hepatocytes (9, 10), was recently identified as a cellular receptor for both HDV and HBV infection (11). The human hepatoma HepG2 cell line complemented with NTCP has become a valuable platform to study viral infections in vitro (12). Cell culture studies indicated that viral infections of HDV and HBV in species other than human are restricted by their NTCPs at entry level (11,13,14). We along with others showed that HDV can infect multiple mammalian cell lines expressing human but not mouse NTCP (13, 15). We were also able to further show that transgenic (Tg) C57BL/6 mice exogenously expressing human NTCP (hNTCP-Tg) in liver support transient HDV infection in vivo, that the infection was acute and age dependent, and that clearance of the viral infection was complex and likely mediated by innate immunity responses (16).Extensive mutagenesis studies using cell cultures have also revealed that residues H84, T86, and S87 in mouse NTCP are responsible for the species restriction for viral infection (13,15). At present, no structural information of NTCP is available. From studying a structurally related protein, apical sodium-dependent bile acid transporter (ASBT), it is speculated that these three residues may locate at the first extracellular loop of NTCP and might be involved in the interaction of the pre-S1 domain of viral L protein with the receptor and mediate subsequent viral entry (14).

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confidence: 55%

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confidence: 99%

Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D Virus In Vivo

Wang

Cao

Mao

et al. 2016

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“…This suggests that NC integrity and uncoating are also subject to host regulation. As normal mouse hepatocytes are unable to form CCC DNA or significant amounts of PF-RC DNA (54,55), possibly due to a deficiency in NC uncoating (54), and are resistant to HBV infection, even after receptor reconstitution (56,57), these results further implicate NC disassembly or uncoating as a critical step in determining HBV species tropism.…”

Section: Discussionmentioning

confidence: 93%

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation

Cui

Luckenbaugh

Bruss

et al. 2015

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCEHepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.T he human pathogen hepatitis B virus (HBV) belongs to the family of Hepadnaviridae, a group of small, hepatotropic DNA viruses that also include closely related animal viruses, such as the duck hepatitis B virus (DHBV) (1). Hepadnaviruses contain a small (ca. 3-kb), partially double-stranded (ds-), relaxed circular (RC) DNA genome enclosed within an icosahedral capsid that is, in turn, formed by multiple copies (240 or 180) of ...

“…Conversely, introduction of NTCP cDNA into HepG2 and Huh7 cells conferred susceptibility to infection by HBV and HDV, respectively (16). These seminal findings established NTCP as an HBV and HDV receptor, a demonstration that has been independently confirmed and extended (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Consequently, NTCP substrates or inhibitors such as tauroursodeoxycholic acid (TUDCA), cyclosporine, irbesartan, and ritonavir could suppress ccHBV or HDV infection (18,(20)(21)(22)(23)(24).…”

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confidence: 89%

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor

Zong

Sureau

et al. 2016

Hepatitis B virus (HBV) is a major etiological agent for liver cirrhosis and hepatocellular carcinoma (1). The nature of the liver-specific HBV receptor(s) has been a longstanding puzzle in the field (2); this is partly attributable to the paucity of cell lines supporting productive HBV infection. Other than primary human hepatocytes (PHHs) and primary tupaia hepatocytes (PTHs), only the human liver progenitor cell line HepaRG could be infected with HBV after prolonged treatment with dimethyl sulfoxide (DMSO) (3). DMSO promotes the differentiation of HepaRG cell into foci of hepatocytes surrounded by biliary cells. Other human hepatoma cell lines such as HepG2 and Huh7 support HBV DNA replication and virion production upon transfection with cloned HBV genome but not after inoculation with HBV particles. HBV protein expression and genome replication are driven by several coterminal transcripts ranging from 0.7 to 3.5 kb (4). The subgenomic RNAs of 2.4 and 2.1 kb are responsible for the expression of three coterminal envelope proteins termed large (L), middle (M), and small (S), with the M protein having an extra preS2 domain than the S protein and the L protein having an extra preS1 domain than the M protein. In addition to their incorporation into virions, the envelope proteins, especially S and M, are secreted as capsid-free subviral particles that exceed virions by Ն1,000-fold. The large quantity of S protein associated with subviral particles is detected by enzyme-linked immunosorbent assay (ELISA) as hepatitis B surface antigen (HBsAg), which provides a sensitive serological marker of HBV infection. Another serological marker is hepatitis B e antigen (HBeAg), a secreted