<i>De novo</i> cloned monkey Ntcps.

<p><i>De novo</i> cloned monkey Ntcps.</p

Ntcp regions critical for HBV/WMHBV binding.

<p><b>(A)</b> PreS1_2-48 peptide sequences of human HBV, genotype D, and of woolly monkey HBV (WMHBV). The following domains are supposed to interact with NTCP/Ntcp: main binding domain (black, amino acids 9–15), accessory binding domains I (dark grey, amino acids 28–39) and II (light grey, amino acids 40–48). <b>(B)</b> Alignment of Ntcps from apes (black), Old World monkeys (orange), and New World monkeys (red). The alignment is restricted to the regions known to be critical for HBV binding (light green, 84–87) and infection (dark green, 157–165). Both regions are highlighted at a homology model of NTCP.</p

Characterisation of the hepatitis B virus cross-species transmission pattern via Na⁺/taurocholate co-transporting polypeptides from 11 New World and Old World primate species

<div><p>The hepatic Na⁺/taurocholate co-transporting polypeptide (NTCP in man, Ntcp in animals) is the high-affinity receptor for the hepatitis B (HBV) and hepatitis D (HDV) viruses. Species barriers for human HBV/HDV within the order Primates were previously attributed to Ntcp sequence variations that disable virus-receptor interaction. However, only a limited number of primate Ntcps have been analysed so far. In the present study, a total of 11 Ntcps from apes, Old and New World monkeys were cloned and expressed <i>in vitro</i> to characterise their interaction with HBV and HDV. All Ntcps showed intact bile salt transport. Human NTCP as well as the Ntcps from the great apes chimpanzee and orangutan showed transport-competing binding of HBV derived myr-preS1-peptides. In contrast, all six Ntcps from the group of Old World monkeys were insensitive to HBV myr-preS1-peptide binding and HBV/HDV infection. This is basically predetermined by the amino acid arginine at position 158 of all studied Old World monkey Ntcps. An exchange from arginine to glycine (as present in humans and great apes) at this position (R158G) alone was sufficient to achieve full transport-competing HBV myr-preS1-peptide binding and susceptibility for HBV/HDV infection. New World monkey Ntcps showed higher sequence heterogeneity, but in two cases with 158G showed transport-competing HBV myr-preS1-peptide binding, and in one case (<i>Saimiri sciureus</i>) even susceptibility for HBV/HDV infection. In conclusion, amino acid position 158 of NTCP/Ntcp is sufficient to discriminate between the HBV/HDV susceptible group of humans and great apes (158G) and the non-susceptible group of Old World monkeys (158R). In the case of the phylogenetically more distant New World monkey Ntcps amino acid 158 plays a significant, but not exclusive role.</p></div

Localization of amino acid 158 at human NTCP.

<p>Homology model of human NTCP based on the crystal structure of ASBT_Yf [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199200#pone.0199200.ref022" target="_blank">22</a>]. The model represents amino acids 27–308 of NTCP. The panel domain is depicted in light and the core domain in dark grey. Localisation of the protein within the plasma membrane is indicated by dashed lines and was calculated based on transmembrane domain predictions performed with HMMTOP and MEMSAT3. Amino acid 158 is highlighted in green. Replacement of glycine 158 with the more bulky arginine abolished myr-preS1-peptide binding and <i>in vitro</i> infection, probably by sterically blocking the virus attachment site of NTCP.</p

HBV/HDV infection via human and monkey NTCP/Ntcps.

<p>HepG2 cells were transiently transfected with human/monkey NTCP/Ntcps wild type or mutant constructs in 96 well plates and inoculated with 10,000 genome equivalents of pseudotyped HDV particles (HDVpsHBV/HDVpsWMHBV) or 2,000 HBV genome equivalents per cell. Cells were cultured for 10 days post infection and then immunostained against HDAg or HBcAg. Infected cells per well were manually counted by fluorescence microscopy. Cell counts are depicted as means ± SD of one representative experiment performed in triplicate.</p

Transport-competing HBV myr-preS1-peptide binding to monkey Ntcps.

<p>HEK293 cells were transiently transfected with human/monkey NTCP/Ntcp wild type or mutant constructs. Transport activity was qualitatively verified with NBD-TC (green fluorescence, nuclei blue fluorescence) and was quantitatively measured with [³H]TC. Absence of myr-preS1 served as positive control (scaled to 100%, open bars). HBV (blue bars) or WMHBV (red bars) myr-preS1-peptides served as inhibitors at increasing concentrations. Negative control: uptake in sodium-free buffer (black bars). Data represent means ± SD of n = 3 determinations. ^#Significant transport inhibition compared to positive control, <i>p</i><0.0001. *Significantly different from the corresponding value of the wild type NTCP/Ntcp, <i>p</i><0.001 (two-way ANOVA).</p

Direct HBV myr-preS1-peptide binding to monkey Ntcps.

<p>Human or monkey NTCP/Ntcp-transfected HEK293 cells were incubated with the fluorescently labelled myr-preS1-HBV-Al633 (blue columns) or myr-preS1-WMHBV-Al633 (red columns) peptides (10 nM, 20 min, 37°C). After washing, cells were analysed for Al633 fluorescence. Data represent means ± SD of three combined independent experiments each with triplicate determinations. Representative fluorescence scans are shown for myr-preS1-HBV-Al633 (left half) and myr-preS1-WMHBV-Al633 (right half). SOAT-expressing HEK293 cells served as negative control. ^*Statistically different from the wild type clone, <i>p</i><0.001. ^#No significant difference to negative control, <i>p</i><0.001 (two-way ANOVA). Rfu, relative fluorescence units.</p

Primers used for <i>de novo</i> cloning of monkey Ntcps.

<p>Primers used for <i>de novo</i> cloning of monkey Ntcps.</p

Primers used for mutagenesis of human NTCP and monkey Ntcps.

<p>Primers used for mutagenesis of human NTCP and monkey Ntcps.</p

Phylogenetic classification of the cloned monkey Ntcps.

<p>Phylogenetic relationship of monkey Ntcps with their habitats in the Old World (orange) or New World (red). Abbreviations, given after the species names, are used instead of the full species names throughout the manuscript. NTCP/Ntcps from non-primate species are included for comparison. Tree-outgroup: <i>Monodelphis domestica</i> (M.dom).</p