Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

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Binding of Nanoparticles Harboring Recombinant Large Surface Protein of Hepatitis B Virus to Scavenger Receptor Class B Type 1

Viruses ◽

10.3390/v13071334 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1334

Author(s):

Shuji Hinuma ◽

Kazuyo Fujita ◽

Shun’ichi Kuroda

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Fluorescent Protein ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Surface Protein ◽

Scavenger Receptor Class ◽

B Virus ◽

Class B

(1) Background: As nanoparticles containing the hepatitis B virus (HBV) large (L) surface protein produced in yeast are expected to be useful as a carrier for targeting hepatocytes, they are also referred to as bio-nanocapsules (BNCs). However, a definitive cell membrane receptor for BNC binding has not yet been identified. (2) Methods: By utilizing fluorescence-labeled BNCs, we examined BNC binding to the scavenger receptor class B type 1 (SR-B1) expressed in HEK293T cells. (3) Results: Analyses employing SR-B1 siRNA and expression of SR-B1 fused with a green fluorescent protein (SR-B1-GFP) indicated that BNCs bind to SR-B1. As mutagenesis induced in the SR-B1 extracellular domain abrogates or attenuates BNC binding and endocytosis via SR-B1 in HEK293T cells, it was suggested that the ligand-binding site of SR-B1 is similar or close among high-density lipoprotein (HDL), silica, liposomes, and BNCs. On the other hand, L protein was suggested to attenuate an interaction between phospholipids and SR-B1. (4) Conclusions: SR-B1 can function as a receptor for binding and endocytosis of BNCs in HEK293T cells. Being expressed various types of cells, it is suggested that functions as a receptor for BNCs not only in HEK293T cells but also in other types of cells.

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P185 PROTEIN KINASE C-INDUCED ENDOCYTOSIS OF SODIUM TAUROCHOLATE COTRANSPORTING POLYPEPTIDE MEDIATES CELLULAR ENTRY OF HEPATITIS B VIRUS IN AN IN VITRO HBV MODEL

Journal of Hepatology ◽

10.1016/s0168-8278(14)60347-0 ◽

2014 ◽

Vol 60 (1) ◽

pp. S127-S128

Author(s):

J.W. Chung ◽

I.Y. Moon ◽

S.W. Yang ◽

E.S. Jang ◽

S.-H. Jeong ◽

...

Keyword(s):

Protein Kinase C ◽

Hepatitis B Virus ◽

Protein Kinase ◽

Hepatitis B ◽

Sodium Taurocholate ◽

Kinase C ◽

B Virus ◽

Cellular Entry ◽

Hbv Model

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Silencing of the scavenger receptor (Class B – Type 1) gene using siRNA-loaded chitosan nanaoparticles in a HepG2 cell model

Colloids and Surfaces B Biointerfaces ◽

10.1016/j.colsurfb.2014.10.045 ◽

2014 ◽

Vol 123 ◽

pp. 930-937 ◽

Cited By ~ 26

Author(s):

Mariane M. Farid ◽

Rania M. Hathout ◽

Mahmoud Fawzy ◽

Khaled Abou-Aisha

Keyword(s):

Hepg2 Cell ◽

Cell Model ◽

Scavenger Receptor ◽

Scavenger Receptor Class ◽

Class B

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Linoleic acid treatment increases the expression of scavenger receptor class B type 1 (SR-B1) in in-vitro model

Atherosclerosis ◽

10.1016/j.atherosclerosis.2021.06.382 ◽

2021 ◽

Vol 331 ◽

pp. e128

Author(s):

N.-A. Azemi ◽

L. Abu-Bakar ◽

N. Ismail ◽

V. Sevakumaran ◽

T.-S. Tengku-Muhammad

Keyword(s):

Linoleic Acid ◽

Acid Treatment ◽

In Vitro Model ◽

Scavenger Receptor ◽

Scavenger Receptor Class ◽

Class B ◽

Vitro Model

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

Journal of Virology ◽

10.1128/jvi.00938-21 ◽

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.

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