E-cadherin binds glycosylated sodium-taurocholate cotransporting polypeptide to facilitate hepatitis B virus entry

Hepatitis B virus (HBV) continues to pose a serious public health risk and is one of the major causes of chronic liver disease and hepatocellular carcinoma. Current antiviral therapy does not effectively eradicate HBV and, thus, further investigation into the mechanisms employed by HBV to allow for invasion of host cells, is critical for the development of novel therapeutic agents. Sodium-taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for HBV. However, the specific mechanism by which HBV and NTCP interact remains unclear. Herein we show that the expression of E-cadherin was upregulated in cells expressing HBV, while knockdown of E-cadherin in HepG2-NTCP cells, HepaRG cells and primary human hepatocytes served to significantly inhibit infection by HBV and HBV pseudotyped particles. Alternatively, exogenous E-cadherin expression was found to significantly enhance HBV uptake by HepaRG cells. Further, mechanistic studies identified glycosylated NTCP localized to the cell membrane via E-cadherin binding, which subsequently allowed for more efficient binding between NTCP and the preS1 of the large HBV surface proteins. E-cadherin was also found to play a key role in establishing and maintaining hepatocyte polarity, which is essential for efficient HBV infection. These observations suggest that E-cadherin facilitates HBV entry through regulation of NTCP distribution and hepatocyte polarity. Author Summary Hepatitis B Virus (HBV) still seriously endangers public health. It is very important to understand the mechanism of HBV invading host cells for developing new therapy target. Sodium-taurocholate cotransporting polypeptide (NTCP) is the key receptor mediating HBV invasion, while other molecules also exhibit important roles in ensuring efficient and productive HBV infection. This study reports that E-cadherin facilitates HBV entry by directly interacting with glycosylated NTCP to mediate its distribution on the hepatocyte membrane and also affects the efficacy of HBV invasion by influncing hepatocyte polarity.

specifically, is a calcium-dependent adhesion integrin that is abundant in epithelial 101 tissues and plays an important role in cell-cell adhesion complexes including 102 desmosomes and adherens junctions [12]. In our study, we found significantly 103 increased expression of E-cadherin in cells that stably or transiently expressed  in HepG2-NTCP and HepaRG cell lines (Fig 2A and 2B). Moreover, silencing of 133 E-cadherin and NTCP significantly reduced the level of HBV 3.5 kb mRNA in 134 HepG2-NTCP, HepaRG and PHH cell lines ( Fig 2C); while silencing of both 135 E-cadherin and NTCP in HepG2-NTCP and PHH cells served to further reduce the 136 level of HBV 3.5 kb mRNA compared to that observed by either E-cadherin or NTCP 137 separately. Further, western blot and immunofluorescence analysis determined that 138 downregulation of E-cadherin or NTCP independently, or together, significantly 139 reduced the expression of HBV core protein and HBV HBsAg with similar inhibition 140 efficiencies (Fig 2D and 2E). These results suggest that silencing of E-cadherin  Silencing of E-cadherin inhibits infection with HBV particles isolated from the 7 144 serum of an HBV carrier 145 To further elucidate the relationship between E-cadherin and HBV infection,

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HepG2-NTCP and PHH cells were infected with HBV particles obtained from a 147 chronic HBV patient. Infection were detected after four days. Silencing of E-cadherin 148 or NTCP alone or at the same time, acted to significantly reduce the level of HBV 3.5 149 kb mRNA in HepG2-NTCP and PHH cells ( Fig 3A). Moreover, silencing of 150 E-cadherin also significantly inhibited the level of HBV core and HBsAg proteins 151 (Fig 3B and 3C). These results suggest that silencing of E-cadherin also inhibits 152 infection by HBV particles isolated from the serum of HBV carriers.

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Overexpression of E-cadherin promotes HBV particles infection 154 The concentration of E-cadherin was determined to be lower in HepaRG cells  E-cadherin overexpression increased the level of HBV 3.5 kb mRNA (Fig 4B). 161 Furthermore, overexpression of E-cadherin served to enhance the protein level of 162 HBV core and HBsAg proteins (Fig 4C and 4D). These results suggest that  HepG2-NTCP and HepaRG cells (Fig 5A and 5B), suggesting that E-cadherin 170 impacts HBV binding/entry rather than HBV replication.

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Silencing of E-cadherin inhibits HBV pre-S1 binding and internalization to 172 HepG2-NTCP and PHH cells 173 We next sought to determine the mechanism employed by E-cadherin to enhance 174 HBV binding. We, therefore, quantified the expression of HBV pre-S1 (Myr-2-47aa) 175 via immunofluorescence assays in HepG2-NTCP and PHH cells after 120 min 176 incubation at 4 °C, which is the conditions in which viral binding most readily occurs, 177 or at 37 °C, when uptake of pre-S1 occurs. Results revealed that when E-cadherin or 178 NTCP were silenced separately or at the same time, significant inhibition of preS1 179 binding and uptake were observed in HepG2-NTCP and PHH cells (Fig 6A and 6B).

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These results suggest that E-cadherin modulates HBV entry by affecting preS1 181 binding and internalization by host cells. 183 To further explore the mechanism by which E-cadherin mediates HBV particle 184 entry, we examined whether it influenced the total intracellular NTCP concentration.

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Results show that silencing of E-cadherin did not affect intracellular NTCP in 186 HepG2-NTCP and HepaRG cells (Fig 7A and 7B), which suggests that E-cadherin 187 does not modulate HBV entry by directly affecting the expression and stability of 9 188 NTCP. We, therefore, speculated that E-cadherin may influence the membrane 189 distribution of NTCP. Our results clearly shown that silencing E-cadherin in  Fig 7F). Furthermore, after treating cell membrane proteins with PNGase F, 206 we also observed a shift in electrophoresis size from 50 kDa to 39 kDa, indicating that 207 a large fraction of the NTCP localized at the cell membrane was glycosylated ( Fig 7F). 208 Lastly, after incubating preS1 with HepG2-NTCP cell lysates, we observed the 209 formation of preS1, glycosylated NTCP and E-cadherin complexes ( Fig 7G). Taken 10 210 together these results suggest that E-cadherin binds to glycosylated NTCP, allowing 211 for efficient localization to the cell surface.   upregulates E-cadherin expression, which acts as a novel host factor that facilitates 246 HBV entry. The functional role of E-cadherin as a host entry factor was confirmed by 247 numerous studies. Specifically we showed that silencing of E-cadherin acted to inhibit 248 HBV particle entry into HepG2-NTCP, HepaRG and PHH cells (Fig 2 and 3); 249 overexpression of E-cadherin contributed to HBV particle entry in HepaRG cells (Fig   250   4) and silencing of E-cadherin inhibited HBVpps entry in HepG2-NTCP and HepaRG 251 cells (Fig 5). Moreover, E-cadherin silencing caused a significant decrease in the 252 binding and internalization of the HBV pre-S1 peptide by HepG2-NTCP and PHH 253 cells (Fig. 6). Mechanistic studies suggested that E-cadherin regulates the cell-surface 12 254 distribution of NTCP (Fig 7) and affects hepatocyte polarization (Fig 8). This study 255 represents an important step forward in understanding the molecular mechanisms and 256 cellular regulatory events involved in HBV entry. 257 Previous studies reported that HBx or HBV inhibit E-cadherin expression. 258 However, contrary to these reports, we have shown that the expression of E-cadherin 259 was upregulated in cells that transiently or stably expressed HBV (Fig 1). and HBsAg) to confirm infection of HepG2-NTCP cells (Fig 3). Similarly, Yan et al. is from 39 kDa to 56 kDa [29]. Surprisingly, we have also discovered that E-cadherin 302 interacts exclusively with the glycosylated form of NTCP (Fig 7E). A previous study 303 indicated that glycosylation is essential for NTCP to act as a receptor for HBV since  Moreover, Myr-preS1 was found to bind exclusively to the glycosylated form (Fig 7). 312 Our results showed that E-cadherin was exclusively associated with glycosylated 313 NTCP (Fig 7B), and, thus, we propose that E-cadherin exerts a regulatory role on the 314 cellular entry of HBV through interacting with glycosylated NTCP and facilitating its 315 membrane localization in hepatocytes.  conditions. ZO-1, located at the edges of the HepG2-NTCP cluster were found to 357 exhibit linear polarity on the cell membrane ( Fig 8D). Importantly, these 358 HepG2-NTCP cells at the edges of the cluster were found to be more susceptible to 359 HBV infection (Fig. 8E). These results suggest that polarized hepatocytes are more 360 susceptible to HBV infection, which is consistent with the results of previous studies.