NTCP oligomerization occurs downstream of the NTCP-EGFR interaction during hepatitis B virus internalization

2021 ◽  
Author(s):  
Kento Fukano ◽  
Mizuki Oshima ◽  
Senko Tsukuda ◽  
Hideki Aizaki ◽  
Mio Ohki ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Bile Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Sodium Taurocholate ◽  
Growth Factor Receptor ◽  
Host Cells ◽  
B Virus ◽  
Epidermal Growth

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and non-competitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, co-immunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affect the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction, and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. Importance Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization, and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry, and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

scholarly journals The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network

2019 ◽  
Vol 295 (3) ◽  
pp. 800-807 ◽  
Author(s):  
Masashi Iwamoto ◽  
Wakana Saso ◽  
Kazane Nishioka ◽  
Hirofumi Ohashi ◽  
Ryuichi Sugiyama ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Hepatitis B Virus ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Hepatitis B ◽  
Viral Infection ◽  
Growth Factor Receptor ◽  
Downstream Signaling ◽  
B Virus ◽  
Epidermal Growth

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

Trans-Activation of Epidermal Growth Factor Receptor Gene by the Hepatitis B Virus X-Gene Product

Virology ◽  
1993 ◽  
Vol 196 (2) ◽  
pp. 878-882 ◽  
Author(s):  
Stefano Menzo ◽  
Massimo Clementi ◽  
Elena Alfani ◽  
Patrizia Bagnarelli ◽  
Silvia Iacovacci ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Hepatitis B Virus ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Hepatitis B ◽  
Gene Product ◽  
Receptor Gene ◽  
Growth Factor Receptor ◽  
B Virus ◽  
Epidermal Growth

scholarly journals Establishment of a novel hepatitis B virus culture system using immortalized human hepatocytes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuichi Akahori ◽  
Hiroki Kato ◽  
Takashi Fujita ◽  
Kohji Moriishi ◽  
Yasuhito Tanaka ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Lines ◽  
Culture System ◽  
Sodium Taurocholate ◽  
Three Dimensional ◽  
Human Hepatocytes ◽  
Host Cells ◽  
B Virus ◽  
Almost All

AbstractRecent development of hepatitis B virus (HBV) culture systems has made it possible to analyze the almost all steps of the viral life cycle. However, the reproducibility of interaction between HBV and host cells seemed inaccurate in those systems because of utilization of cancer cell lines with a difference from hepatocytes in the majority of cases. In this study, in order to resolve this point, a novel HBV culture system using non-cancer-derived immortalized human hepatocytes derived cell lines, producing exogenous human sodium taurocholate cotransporting polypeptide, was developed. One of the cell clones, E/NtG8 cells, was permissive to both blood-borne HBV (HBVbb) and culture-derived recombinant HBV when cultured in the three-dimensional condition. Furthermore, the production of infectious HBV particles, which showed the similar physicochemical properties to HBVbb, was observed for about a month after HBVbb infection in this system, suggesting that it may reproduce whole steps of the HBV lifecycle under the condition analogous to human liver cells infected with HBV. This system seemed to contribute not only to find novel interactions between HBV and host cells but also to understand mechanism of HBV pathogenesis.

scholarly journals Curcumin inhibited hepatitis B viral entry through NTCP binding

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Piyanoot Thongsri ◽  
Yongyut Pewkliang ◽  
Suparerk Borwornpinyo ◽  
Adisak Wongkajornsilp ◽  
Suradej Hongeng ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Viral Entry ◽  
Sodium Taurocholate ◽  
Hbv Dna ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Hepatic Cells ◽  
B Virus ◽  
Site Of Action ◽  
Uptake Assay

AbstractHepatitis B virus (HBV) has been implicated in hepatitis and hepatocellular carcinoma. Current agents (nucleos(t)ide analogs and interferons) could only attenuate HBV infection. A combination of agents targeting different stages of viral life cycle (e.g., entry, replication, and cccDNA stability) was expected to eradicate the infection. Curcumin (CCM) was investigated for inhibitory action toward HBV attachment and internalization. Immortalized hepatocyte-like cells (imHCs), HepaRG and non-hepatic cells served as host cells for binding study with CCM. CCM decreased viral load, HBeAg, HBcAg (infectivity), intracellular HBV DNA, and cccDNA levels. The CCM-induced suppression of HBV entry was directly correlated with the density of sodium-taurocholate co-transporting polypeptide (NTCP), a known host receptor for HBV entry. The site of action of CCM was confirmed using TCA uptake assay. The affinity between CCM and NTCP was measured using isothermal titration calorimetry (ITC). These results demonstrated that CCM interrupted HBV entry and would therefore suppress HBV re-infection.

Evidence for Dual Effects of DNA-Reactive Bile Acid Derivatives (Bamets) on Hepatitis B Virus Life Cycle in an In Vitro Replicative System

2002 ◽  
Vol 13 (6) ◽  
pp. 371-380 ◽  
Author(s):  
Marta R Romero ◽  
Maria C Martinez-Diez ◽  
Monica G Larena ◽  
Rocio IR MacIas ◽  
Mercedes Dominguez ◽  
...  
Keyword(s):  
Life Cycle ◽  
Bile Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Dna ◽  
Drug Uptake ◽  
Host Cells ◽  
B Virus ◽  
Bile Acid Derivatives ◽  
Hepg2 2.2.15

A liver targeting strategy to direct antiviral drugs toward hepatitis B virus (HBV) was investigated. As model drugs we used cisplatin-bile acid derivatives (Bamets) to determine the production of virions by HBV-transfected hepatoblastoma cells (HepG2 2.2.15). Drug uptake was determined using flameless atomic absorption spectrometry to measure platinum cell contents. Cytotoxic effect was determined by formazan formation and neutral red uptake tests. The release of viral surface protein was evaluated by ELISA. The abundance of HBV-DNA in the medium was determined by quantitative real-time PCR and its structure by Southern blot analysis. The uptake of Bamets by HepG2 2.2.15 cells was higher than that of cisplatin. At concentrations lower than 10 μM, distinct Bamets have no toxic effect on host cells, whereas cisplatin dramatically reduced cell viability at concentrations higher than 1 μM. All the drugs tested inhibited the release of viral proteins to the medium, but induced a marked and progressive dose-dependent increase in the amount of viral DNA in the medium. This was mainly due to the release of short fragments of HBV-DNA in the case of cisplatin. On the contrary, Bamets induced an enhanced release of circular forms of HBV-DNA. These findings suggest the existence of a dual effect of Bamets on HBV life-cycle by enhancing the production of DNA replicative intermediates but reducing the secretion of complete virions. Altogether these characteristics recommend consideration of these compounds as a useful experimental tool in the investigation of novel liver targeted therapeutic agents based on bile acid derivatives for the treatment of HBV infections, or to carry out further studies on the HBV life cycle.

scholarly journals Hepatitis B Virus-Encoded X Protein Downregulates EGFR Expression via Inducing MicroRNA-7 in Hepatocellular Carcinoma Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Yun-Ju Chen ◽  
Pei-Hsuan Chien ◽  
Wen-Shu Chen ◽  
Yu-Fong Chien ◽  
Ya-Ying Hsu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Growth Rate ◽  
Hepatitis B Virus ◽  
Cell Growth ◽  
Hepatitis B ◽  
Growth Factor Receptor ◽  
Egfr Expression ◽  
B Virus ◽  
Epidermal Growth ◽  
Hcc Cells

Hepatitis B virus (HBV) infection accounts for over a half of cases of hepatocellular carcinoma (HCC), the most frequent malignant tumor of the liver. HBV-encoded X (HBx) plays critical roles in HBV-associated hepatocarcinogenesis. However, it is unclear whether and how HBx regulates the expression of epidermal growth factor receptor (EGFR), an important gene for cell growth. Therefore, the study aimed to investigate the association between HBx and EGFR expression. In this study, we found that HBx upregulates miR-7 expression to target 3′UTR of EGFR mRNA, which in turn results in the reduction of EGFR protein expression in HCC cells. HBx-mediated EGFR suppression renders HCC cells a slow-growth behavior. Deprivation of HBx or miR-7 expression or restoration of EGFR expression can increase the growth rate of HCC cells. Our data showed the miR-7-dependent EGFR suppression by HBx, supporting an inhibitory role of HBx in the cell growth of HCC. These findings not only identify miR-7 as a novel regulatory target of HBx, but also suggest HBx-miR-7-EGFR as a critical signaling in controlling the growth rate of HCC cells.

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

2019 ◽  
Author(s):  
Qin Hu ◽  
Fei-Fei Zhang ◽  
Liang Duan ◽  
Bo Wang ◽  
Pu Li ◽  
...  
Keyword(s):  
Public Health ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Host Cells ◽  
Heparg Cells ◽  
B Virus ◽  
Hepatocyte Polarity ◽  
E Cadherin

AbstractHepatitis 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 SummaryHepatitis 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.

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