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

Abstract

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. Hepatitis B virus (HBV) is the prototype of the Hepadnaviridae (hepatotropic DNA viruses) family (1). Human HBV has infected 2 billion people worldwide, and 350 million of them are chronically infected (2). About two-thirds of hepatocellular carcinoma (HCC) is due to chronic HBV infection (3). Hepatitis D virus (HDV) is a satellite virus of HBV, 15 million people are infected by HDV, and no specific anti-HDV drug is clinically available at present. Chronic HBV patients coinfected with HDV are at high risk for more severe symptoms and more rapid progression (4).HBV is a small enveloped virus with a relaxed circular partially double-stranded DNA genome of ϳ3.2 kb encoding four overlapped open reading frames. HBV large (L), middle (M), and small (S) envelope proteins are encoded by a single open reading frame (5). They are translated from different initial codons but share an end. HDV contains a single-stranded, circular RNA genome of ϳ1,700 nucleotides, with one coding region for small and large form of delta antigens. It replicates in the nucleus and accumulates a large number of viral RNAs and delta antigen (6). Since HDV has to employ HBV envelope proteins for the infection of hepatocytes (7), the entry of HDV is believed to be similar to that of HBV and has been used as a surrogate to study the early entry process (4,8,9).The lack of a convenient in vitro viral infection system has been a long-standing hurdle for studying viral entry of HBV and HDV (10). Recently, ...

Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting Polypeptide

“…1B). These results were consistent with our previously published results indicating that the pre-S1 lipopeptide can bind to hNTCP, mNTCP, and mkNTCP-h157-165 but not wild-type mkNTCP or hNTCPmk157-165 (3,5). Interestingly, the same peptide with a cholesterol modification at the N-terminal amino acid could also inhibit [ 3 H]taurocholate uptake in HepG2-NTCP in a dose-dependent manner (Fig.…”

“…Multiple lines of evidence support that NTCP is likely a dominant receptor for HBV and HDV (3)(4)(5). The expression of NTCP correlates with the susceptibility of the target cells, and reducing the expression of NTCP markedly inhibits HBV and HDV infection on known susceptible cells.…”

Association of Genetic Variation of Sodium Taurocholate Cotransporting Polypeptide with Chronic Hepatitis B Virus Infection

“…Receptors confer the cell permissiveness to the virus and also serve as necessities for the virus transmission between cells, probably explaining the predisposing effect of the SNP in the receptor NTCP gene on both the susceptibility and chronicity in HBV infection, especially the susceptibility. It should be noted that the polymorphism genotyped in this study appears to be not located within the amino acids 157 to 165 of NTCP, which have been demonstrated to be critical for viral entry of HBV (Yan et al, 2012(Yan et al, , 2013. Whether it is conformationally and/or functionally important for influencing the viral entry awaits to be clarified.…”

Current and future directions for treating hepatitis B virus infection

“…Sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor [118,119] . Iwamoto et al [120] , Watashi et al [121] and Tsukuda et al [122] reported that cyclosporine A and its analogs blocked HBV entry through inhibiting the interaction between NTCP and the HBV large surface protein.…”