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University of Birmingham Lentiviral Hepatitis B Pseudotype Entry University of Birmingham Lentiviral hepatitis B pseudotype entry requires NTCP and additional hepatocyte-specific factors Meredith, L W; Hu, K; Cheng, X; Howard, C R; Baumert, T F; Balfe, P; van de Graaf, K F; Protzer, U; McKeating, J A DOI: 10.1099/jgv.0.000317 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Meredith, LW, Hu, K, Cheng, X, Howard, CR, Baumert, TF, Balfe, P, van de Graaf, KF, Protzer, U & McKeating, JA 2016, 'Lentiviral hepatitis B pseudotype entry requires NTCP and additional hepatocyte-specific factors', Journal of General Virology, vol. 97, no. 1, pp. 121-127. https://doi.org/10.1099/jgv.0.000317 Link to publication on Research at Birmingham portal General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact [email protected] providing details and we will remove access to the work immediately and investigate. Download date: 24. Sep. 2021 Journal of General Virology (2016), 97, 121–127 DOI 10.1099/jgv.0.000317 Short Lentiviral hepatitis B pseudotype entry requires Communication sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors L. W. Meredith,13 K. Hu,13 X. Cheng,2 C. R. Howard,1 T. F. Baumert,3 P. Balfe,1 K. F. van de Graaf,4 U. Protzer2,5 and J. A. McKeating1,6 Correspondence 1Centre for Human Virology, Viral Hepatitis Laboratory, College of Medical and Dental Sciences, Jane A. McKeating University of Birmingham, Birmingham, UK [email protected] 2Institute of Virology, Technische Universita¨t Mu¨nchen/Helmholtz Zentrum Mu¨nchen, Munich, Germany 3INSERM U1110, Institut de Recherche sur les Maladies Virales et He´patiques and Universite´ de Strasbourg, Strasbourg, France 4Tytgat Institute for Liver and Intestinal Research and Department of Gastroenterology and Hepatology, AMC, Amsterdam, The Netherlands 5German Center for Infection Research (DZIF), Munich, Germany 6Institute for Advanced Study, Technische Universita¨t Mu¨nchen, Garching, Germany Hepatitis B virus (HBV) is one of the world’s major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. Received 19 June 2015 These results highlight the value of the HBVpp system to dissect the pathways of HBV entry Accepted 16 October 2015 and dissemination. Hepatitis B virus (HBV) infection is a major global health and antigen expression in the liver. HBV infectivity is problem frequently resulting in progressive, degenerative dependent on the large (L) and surface (S) envelope liver disease, including cirrhosis and hepatocellular carcin- glycoproteins, with essential domains mapping to the oma. Despite the availability of a safe and effective vaccine, N-terminal 75 aa (Pre-S1) termed the ‘receptor binding and a range of nucleoside analogue antivirals, ,240 million domain’ and to the antigenic ‘a’ loop of S (reviewed by people are chronically infected with HBV (El-Serag, 2012). Baumert et al., 2014). A myristoylated peptide correspond- The high rate of viral turnover has resulted in vaccine- ing to the N-terminal Pre-S1 region, Myrcludex B, inhibits related escape mutants and drug resistance (Billioud et al., HBV infection (Volz et al., 2013) and was used as bait to 2012; Locarnini & Yuen, 2010). Therefore, development of identify its host interacting partner as sodium taurocholate safe, efficient antiviral strategies remains a key challenge co-transporting polypeptide (NTCP) (Ni et al., 2014; Yan for the treatment of HBV. et al., 2012). NTCP (SLC10A1) is the founding member of the SLC10 family of solute carrier proteins and is exclu- A major obstacle to discovering new therapies is the lack of sively expressed in hepatocytes, where it plays an important efficient in vitro systems or small animal models supporting role in bile acid uptake. HBV infection (Thomas & Zoulim, 2012). Our limited knowledge of the host pathways regulating HBV entry Although exogenous expression of NTCP in human hepa- and dissemination hinders the development of model toma cells renders them susceptible to HBV infection, high systems that recapitulate the dynamics of HBV infection levels of viral inocula (100 genome copies per cell) and polyethylene glycol are required to initiate replication 3These authors contributed equally to this paper. (Ni et al., 2014; Yan et al., 2012). As these infections require Downloaded from www.microbiologyresearch.org by 000317 G 2015 The Authors Printed in Great Britain 121 IP: 147.188.224.127 This is an Open Access article distributed under the terms of the creative commons attribution License (http://creativecommons.org/licenses/by/3.0/). On: Mon, 25 Jul 2016 11:38:42 L. W. Meredith and others (a) Pseudovirus Co-transfection production Infection HBV-L or HBV H.13 – – pNL43-luc-R E Target cell lysed (HIV gag/pd+luc) and luminescence analysed (b) Huh-7 Huh-7.NTCP 293T Pre-S1 (c) (d) (e) (f) *** ** 1.5 15 ** 250 200 * 200 ) ) 150 4 1.0 10 4 ) ) –1 150 405 100 p24 Infectivity HBsAg (OD 100 (RLU x10 Virus entry (pg ml 0.5 5 (RLU x10 50 50 0.0 0.0 0 0 A D C 293T 293T 293T Huh-7 Huh-7 Huh-7 Huh-7.NTCP Huh-7.NTCP Huh-7.NTCP (g) HBsAg p24 HBVpp VSVpp 0.6 24 4 200 Viral entry (RLU x10 ) 3 3 150 (pg ml VSVpp ) 22 0.4 p24 405 –1 2 100 HBVpp HBsAg ) (OD 0.2 20 1 50 3 ) Viral entry (RLU x10 0.0 18 0 0 0 0 30 26 22 18 14 10 30 26 22 18 14 10 Fraction (% iodixanol) Fraction (% iodixanol) 2 2 Fig. 1. HBV pseudovirus genesis. (a) HBVpps are produced by co-transfecting pNL4.3-luc-R E , a lentiviral HIV expression vector, with replication-competent HBV genome H1.3 (genotype D) into parental Huh-7, NTCP-expressing hepatoma cells or 293T embryonal kidney cells. (b) The cells were cultured for 72 h, stained with mAb 18/7, specific for Pre-S1, and imaged by confocal microscopy. Bar, 20 mm. (c, d) Extracellular expression of HBsAg or HIV p24 was quantified by ELISA. (e) To evaluate HBV pseudovirus infectivity, the extracellular media (normalized for p24 content) was allowed to infect dHepG2.NTCP cells for 72 h and luciferase activity measured. (f) Huh-7.NTCP cells were co-transfected with HBV genotype 2 2 A, C or D replication-competent genomes and pNL4.3-luc- R E ; 72 h later the extracellular media was assessed for Downloaded from www.microbiologyresearch.org by 122 Journal of General Virology 97 IP: 147.188.224.127 On: Mon, 25 Jul 2016 11:38:42 HBV entry requires NTCP and additional hepatocyte factors infection of dHepG2.NTCP cells. (g) HBVpps or VSVpps were separated by density ultracentrifugation for 24 h at 100 000 g at 4 8C, across a 10–30 % iodixanol gradient, and each fraction assessed for HBsAg and p24 by ELISA or infec- tion of dHepG2.NTCP cells. Results are the mean¡SD of three independent experiments. The no envelope control [500– 800 relative light units (RLU)] value was subtracted from the HBVpp RLU value and differences assessed by Student’s t-test (*P,0.05, **P,0.01, ***P,0.001). 5–7 days to generate detectable antigen or viral genomes, used to infect DMSO-differentiated HepG2.NTCP cells 2 2 the current culture systems limit studies on HBV entry. (dHepG2.NTCP). HBV H1.3 and pNL4.3-luc-R E plas- mids were also transfected into human embryonal kidney In order to develop a robust methodology to quantify HBV 293T cells: this cell type is commonly used for pseudoparti- entry we characterized lentivirus-based pseudoparticles cle genesis due to its high transfection efficiency; however, it (HBVpps). This system has been used to study the entry does not support HBV replication (Reese et al.,2013;Tang pathways of a range of viruses, including human immuno- et al., 2001).
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