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Lentiviral 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 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

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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- , 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 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

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(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

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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). All of the transfected cells expressed comparable deficiency virus (HIV), virus (HCV), patho- levels of HBV Pre-S1 (Fig. 1b). Cellular processing and genic avian influenza and Ebola, offering a rapid and secretion of HBV surface antigen-containing particles reproducible analysis of the viral –receptor (HBsAg) and HIV p24 was confirmed by interactions (Moller-Tank & Maury, 2015; Temperton ELISA (Fig. 1c, d). We observed significantly higher levels of et al., 2007; Wright et al., 2009; Zimmer et al., 2014). Pre- HBsAg secretion from 293T compared with Huh-7 and vious reports showed that lentiviral HBVpps were infec- Huh-7.NTCP cells. However, comparable levels of extracellular tious for human hepatocytes; however, given the limited HIV p24 were detected for all three cell lines (Fig. 1d), consist- knowledge of internalization pathways at the time, the ent with similar levels of lentiviral core secretion. receptor dependency was not studied (Chai et al., 2007; Sung & Lai, 2002). Our studies show that infectious Having established that both lentiviral capsid and HBV HBVpp genesis is optimal in hepatoma cells, suggesting particles are secreted, we measured the infectivity of pseudo- that liver-specific factors are required for assembly and/or virus preparations in dHepG2.NTCP cells. HBVpps produced infectivity. HBVpp entry is NTCP-dependent; however, by hepatoma cells were 30- to 250-fold more infectious than expression of NTCP in non-liver epithelial cells does not pseudovirus secreted from 293T cells (Fig. 1e), suggesting that confer permissivity to infection, suggesting that additional hepatocyte-specific factors play a role in the assembly and/or hepatocyte-specific factors are required. The recent report secretion of infectious particles. All pseudotype preparations of a genetic polymorphism (p.Ser267Phe) in NTCP that were normalized for HIV p24 content for infection studies ablates HepG2 ability to support HBV infection in vitro and infectivity values represent the net luciferase activity and yet supports HBV infection in man (Peng et al., where the empty control virus activity is subtracted. Interest- 2015) suggests that additional host factors are required ingly, we noted that HBVpps produced from Huh-7.NTCP for HBV entry. Taken together, these results highlight the cells were eightfold more infectious than virus generated by utility of the pseudovirus approach to dissect the molecular parental Huh-7 cells (Fig. 1e). HBV Pre-S1 engagement of pathways of HBV entry into the liver. NTCP has been reported to alter the expression of genes Whilst there are several methods of generating pseudo- involved in bile acid metabolism (Oehler et al., 2014), and viruses, we chose a luciferase-based HIV lentiviral system these pathways may play a role in promoting the assembly 2 2 (pNL4.3-luc-R E ), similar to that reported for HCV and/or secretion of pseudoparticles. (Hsu et al., 2003) (Fig. 1a). For our initial studies we gener- To investigate whether pseudoviruses can be generated ated lentiviral pseudotypes using replication competent expressing glycoproteins from diverse HBV genotypes we HBV genomes to ensure that all three components of the co-transfected replication-competent HBV clones of geno- 2 2 HBV surface proteins were expressed in a ratio and confor- type A, C and D with pNL4.3-luc-R E into Huh-7.NTCP mation consistent with a native infection. Recent studies and the extracellular virus used to infect dHepG2.NTCP. show that both HepG2 and Huh-7 cells transduced to All three genotypes expressed similar levels of HBsAg and express NTCP support HBV replication (Ni et al.,2014; generated infectious pseudovirus; however, genotype D Yan et al., 2012); however, due to the low transfection effi- was significantly more infectious than the other clones ciency observed with HepG2 cells we selected Huh-7 cells to tested (Fig. 1f). This observation suggests that increased generate lentiviral pseudoparticles. Parental Huh-7 or Huh- entry efficiency contributes to the enhanced infectivity of 7.NTCP cells were co-transfected with equal ratios of lenti- 2 2 genotype D virus observed in vitro. virus pNL4.3-luc-R E and replication-competent 1.3-fold overlength HBV (H1.3) (genotype D) or a control ‘empty’ To assess whether HBVpps are assembled we analysed viral plasmid. The extracellular media collected after 72 h were preparations by gradient centrifugation, and determined

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(a)

HepG2 HepG2.NTCP HepG2 HepG2.NTCP

(c) (b) HepG2 HepG2.NTCP HepG2 HepG2.NTCP 250 20000 ** ** ** *** 200 150000 79% 60% 20% 15% ) 3 150 100000 100 (RLU) Viral entry Viral entry (RLU x10 68% 53% 10% 12% 50000 50

0 0 1:10 1:10 H1.3 1:100 1:100 HBV-L 1:1000 1:1000 1:10000 1:10000 Untreated Untreated

(d) HepG2 HepG2.NTCP (e) HepG2 HepG2.NTCP ** ** *** 200000 ** *** 40000 * ***

150000 30000

100000 20000 (RLU) (RLU) Viral entry Viral entry

50000 10000

0 0 Hepatect ––+ – + – + + – + – + Myr B Myr B lg+Myr lg+Myr HBV lg Genotype A C D A C D HBV lg Control lg Control lg

Fig. 2. HBVpp entry is NTCP- and HBs-dependent. (a) HepG2 or HepG2.NTCP cells were tested for NTCP expression by K9 anti-NTCP Western blotting or confocal microscopy. Bar, 10 mm. (b) Huh-7.NTCP cells were co-transfected with 2 2 replication-competent HBV genotype D or pcDNA3.1 encoding HBV-L and pNL4.3-luc-R E ; 72 h later the extracellular media were collected and assessed for infection of dHepG2 or dHepG2.NTCP cells. (c) Pseudoparticles generated from 2 2 HBV-L/pNL4.3-luc-R E transfected Huh-7.NTCP producer cells were incubated with increasing doses of anti-HBs pooled IgG or control irrelevant IgG for 1 h, before infecting dHepG2 or dHepG2.NTCP cells for 72 h; anti-HBs IgG inhibition (%) 2 2 is shown. (d) Pseudoparticles generated from HBV genotype A, C or D/pNL4.3-luc-R E co-transfected Huh-7.NTCP

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producer cells were incubated with anti-HBs (1 : 100 dilution) or control IgG for 1 h prior to infecting dHepG2 or dHepG2.NTCP cells for 72 h. (e) dHepG2 or HepG2.NTCP were treated with Myrcludex B (Myr B) (1 mM), anti-HBs immunoglobulin (Ig) (1 : 10 dilution) or irrelevant immunoglobulin for 1 h before infecting with HBVpp genotype D for 72 h before quantifying infection. Results are the mean¡SD of at least three experiments, with differences assessed using Student’s t-test (*P,0.05, **P,0.01, ***P,0.001).

whether HBsAg and lentiviral p24 locate in the same frac- 1 mM Myrcludex B, Hepatect (1 : 10) or an irrelevant tion. HBVpps or control pseudoparticles expressing vesicu- immunoglobulin, or a combination of both for 1 h prior lar stomatitis virus G (VSVpp) generated from to infecting with HBVpps or VSVpps. Myrcludex signifi- Huh-7.NTCP cells were fractionated over 10–30 % cantly inhibited HBVpp infection of dHepG2.NTCP and iodixanol gradients by ultracentrifugation at 100 000 g we noted an additive effect of combining Myrcludex with for 24 h at 4 8C. Each fraction was assessed for HBsAg/ Hepatect to neutralize HBVpp infection (Fig. 2e). Myrclu- p24 by ELISA and for infectivity in dHepG2.NTCP cells. dex or Hepatect had no effect on VSVpp infection (data HBsAg and p24 were detected in the same fractions, con- not shown). sistent with the secretion of intact pseudoparticles and To investigate whether HBVpps show a restricted tropism infectivity of HBVpps and control VSVpps associated for liver-derived cells we measured their ability to infect with fractions between 18 and 22 % iodixanol (Fig. 1g), three liver-derived cell lines (Huh-7, HepG2 and Hep3B) demonstrating the assembly of HBs-containing lentiviral and two non-liver epithelial cell lines [293T (embryonal pseudoparticles. kidney) and A549 (lung epithelial)] in the presence or We assessed the ability of HBVpps to infect parental absence of exogenous NTCP expression. As a control, we HepG2 (NTCP null) and those transduced to express included pseudoparticles that could only infect hepatocytes NTCP, confirmed by quantitative real-time PCR measure- (HCVpps) or showed broad-range tropism (VSVpps). All ment of NTCP mRNA, anti-NTCP staining by Western cell lines were confirmed to express NTCP by binding flu- blot or confocal microscopy (Fig. 2a) and bile salt uptake orescent-labelled Myrcludex (Fig. 3a) or K9 anti-NTCP (data not shown). NTCP expression significantly enhanced staining (Fig. 3b). HBVpps only infected the hepatoma HBVpp infection (Fig. 2b), demonstrating common lines and NTCP expression increased entry in all cases receptor-dependent pathways with native HBV. (Fig. 3c). In contrast, HBVpps did not infect 293T or A549 cells in the presence or absence of NTCP, suggesting A significant caveat of generating HBVpps using replica- that NTCP expression alone is not sufficient to confer tion-competent genomes is the concomitant production HBVpp entry and that additional hepatocyte specific fac- of native HBV that will compete with pseudoparticles for tors are required. HCVpps only infected Huh-7 and receptors and reduce reporter gene expression. Hep3B cells, as previously reported (Hsu et al., 2003), We therefore generated pseudovirus by co-transfecting and NTCP expression had no effect (Fig. 3b). pcDNA3.1 encoding HBV L with the lentiviral vector and As expected given its broad tropism, VSVpps infected all observed similar levels of infection to those generated cells types with high efficiency in an NTCP-independent with full-length HBV (Fig. 2b). To confirm that pseudo- manner, demonstrating efficient HIV LTR-driven reporter particle infection was dependent on HBV glycoprotein activity in all cell lines studied. expression we investigated the ability of a clinical-grade polyclonal IgG pooled from vaccinated donors (Hepatect) In conclusion, we have optimized the genesis of infectious to neutralize infection. HBVpps were incubated for 1 h HBVpps, enabling quantitative studies on HBV entry that with increasing doses of Hepatect or an irrelevant IgG, are independent of downstream HBV replication events. before inoculating dHepG2 or dHepG2.NTCP cells. The HBV surface glycoprotein is unlike other viral glyco- We observed a dose-dependent inhibition of HBVpp infec- proteins tested to date (Moller-Tank & Maury, 2015; tion (Fig. 2c), demonstrating that HBVpp infection is HBs- Temperton et al., 2007; Wright et al., 2009; Zimmer dependent. The low-level HBVpp infection observed in par- et al., 2014) in its failure to produce infectious pseudo- ental HepG2 cells was neutralized by Hepatect, suggesting particles in human embryonal kidney cells (293T), requir- an alternative NTCP-independent route for pseudoparticle ing a liver cell background for infectious particles genesis. entry. Hepatect also neutralized the infectivity of HBVpps Our results show that whilst NTCP does play an essential expressing genotype A- and C-encoded glycoproteins role in HBVpp entry, it is unlikely to be the sole receptor (Fig. 2d), confirming HBs-dependent routes of infection facilitating infection. The recent study by Verrier et al. for all pseudotypes. (2015) highlighting a role for Glypican 5, a heparin sul- phate moiety that is highly expressed in the liver, in To confirm the role of NTCP in HBVpp entry, we assessed HBV and HDV infection supports our conclusions. the ability of Myrcludex B to inhibit pseudoparticle infect- Taken together, these results highlight the value of the ivity. dHepG2 or dHepG2.NTCP cells were incubated with HBVpp system to dissect the pathways of HBV entry

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(a) Huh-7 HepG2 Hep3B 293T A549 NTCP Control

(b)

600 Control NTCP

400 (MFI)

NTCP expression NTCP 200

0 293T A549 Huh-7 Hep3B HepG2

(c) HBVpp HCVpp 400000 ** *** *** VSVpp 7×107 300000 5×107 3×107 200000 1×107 (RLU) 5 Viral entry 4×10 100000 (RLU) 5

Viral entry 3×10 2×105 5 0 1×10 0 293T 293T A549 A549 293T Huh-7 A549 Huh-7 Huh-7 Hep3B Hep3B HepG2 HepG2 Hep3B HepG2 293T.NTCP A549.NTCP 293T.NTCP 293T.NTCP Huh-7.NTCP A549.NTCP A549.NTCP Hep3B.NTCP HepG2.NTCP Huh-7.NTCP Huh-7.NTCP Hep3B.NTCP Hep3B.NTCP HepG2.NTCP HepG2.NTCP

Hepatic Non-hepatic Hepatic Non-hepatic Hepatic Non-hepatic

Fig. 3. NTCP expression alone is not sufficient to confer HBVpp infection of non-hepatic cells. (a) Hepatocyte-derived (Huh-7, HepG2, Hep3B) and non-liver embryonal kidney (293T) or lung (A549) epithelial cells were transduced to express NTCP and expression was confirmed by staining with fluorescent-labelled HBV Pre-S1 peptide. (b) NTCP expression was quantified by flow cytometry using K9 anti-NTCP and irrelevant control immunoglobulin, where the data are expressed as mean fluorescence intensity (MFI) of K9 staining and the irrelevant immunoglobulin value subtracted. (c) Parental and NTCP- 2 2 expressing cell lines were infected with HBVpp generated from HBV-L and pNL4.3-luc-R E transfected Huh-7.NTCP cells,

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HCVpps or VSVpps for 72 h. Results are the mean¡SD of three independent experiments where the no envelope RLU value was subtracted from the HBVpp, HCVpp or VSVpp values and significance assessed using Student’s t-test (**P,0.01, ***P,0.001).

and dissemination, and will aid in the discovery of new Oehler, N., Volz, T., Bhadra, O. D., Kah, J., Allweiss, L., Giersch, K., antiviral agents. Bierwolf, J., Riecken, K., Pollok, J. M. & other authors (2014). Binding of hepatitis B virus to its cellular receptor alters the expression profile of genes of bile acid metabolism. Hepatology 60, 1483–1493. Acknowledgements Peng, L., Zhao, Q., Li, Q., Li, M., Li, C., Xu, T., Jing, X., Zhu, X., Wang, Y. & other authors (2015). The p.Ser267Phe variant in SLC10A1 is We would like to thank Bruno Stieger for K9 anti-NTCP and Stefan associated with resistance to chronic hepatitis B. Hepatology 61, Urban for Myrcludex B. Research is supported by the National Insti- 1251–1260. tute for Health Research Birmingham Liver Biomedical Research Unit Reese, V. C., Oropeza, C. E. & McLachlan, A. (2013). Independent (J. M.), Medical Research Council [G1100247 (J. 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