Table 1.
Primers used for de novo cloning of monkey Ntcps.
Table 2.
Primers used for mutagenesis of human NTCP and monkey Ntcps.
Table 3.
De novo cloned monkey Ntcps.
Fig 1.
Phylogenetic classification of the cloned monkey Ntcps.
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: Monodelphis domestica (M.dom).
Fig 2.
Ntcp regions critical for HBV/WMHBV binding.
(A) PreS12-48 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) 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.
Fig 3.
Transport-competing HBV myr-preS1-peptide binding to monkey Ntcps.
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, p<0.0001. *Significantly different from the corresponding value of the wild type NTCP/Ntcp, p<0.001 (two-way ANOVA).
Fig 4.
Direct HBV myr-preS1-peptide binding to monkey Ntcps.
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, p<0.001. #No significant difference to negative control, p<0.001 (two-way ANOVA). Rfu, relative fluorescence units.
Fig 5.
HBV/HDV infection via human and monkey NTCP/Ntcps.
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.
Fig 6.
Localization of amino acid 158 at human NTCP.
Homology model of human NTCP based on the crystal structure of ASBTYf [22]. 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 in vitro infection, probably by sterically blocking the virus attachment site of NTCP.