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41

Fig. 1. HBV infection and life cycle

In the liver, the circulating HBV passes through the fenestrae of LSECs and reaches the space of

Dissé; initiates the cell attachment with HSPG on the cell surface. NTCP, which is exclusively sorted

to the basolateral membrane, has an interaction with PreS1 domain of Large envelope protein on

HBV. EGFR, associates with NTCP, facilitates HBV internalization by EGF stimulation. After virus

entry, it establishes a nuclear pool of episomal DNA into the form of cccDNA, which is copied into

RNA that is transported to the cytoplasm where it reverse-transcribes into DNA and packed in a

virus particle. Exogenous PreS1 peptide can work as a competitive inhibitor and affect HBV

infection.

42

Fig. 2. The development of liver

Liver development begins at mouse embryonic day 9. The cells of the ventral foregut endoderm

are induced to the hepatoblast stage by FGF and BMP signaling from the heart and septum

transversum mesenchyme (STM). Following induction, hepatoblasts proliferate and migrate into

the STM to form the liver bud with non-parenchymal cells, such as endothelial progenitor cells

and hepatic mesenchymal cells. Finally, they differentiate into mature hepatocytes and

cholangiocytes through interactions with LSECs and HSCs. (Castillon et al., 2003).

43

TK

independent

TK

dependent

TK

dependent

Fig. 3. EGFR endocytosis depends on the dose of EGF

EGFR is one of the best-characterized receptor tyrosine kinases (RTKs) and activated by several

ligands, of which EGF is most extensively studied. In general, EGFR is present as the monomeric

inactive form in the absence of EGF and is activated by EGF to form the dimer form. A primary

mechanism of internalization of EGFR is clathrin-mediated endocytosis (CME); the receptor is

removed from the surface as clathrin-coated pits and routed to the early endosome, then recycled

back to the cell surface at a low dose of EGF stimulation. On the other hand, once the receptor is

activated by a high dose of EGF, EGFR is internalized through clathrin-independent endocytosis

(CIE), which is regulated by the phosphorylation level at the C-terminal and ubiquitinylated; after

late endosomal vesicles are trafficked to lysosome to be degradated.

44

Fig. 4. Development of an HBV infection system using iPSC-derived hepatocytes

(A) procedure to induce iPS-hepatocytes. iPSCs were maintained in mono-layer culture on

Matrigel. To initiate definitive endoderm differentiation, iPSCs were cultured in RPMI media

containing B27 supplements and 100 ng/mL activin A. After 5 days of culture under 20%O2, cells

were next moved to 4%O2/5%CO2 in RPMI/B27 media supplemented with 20 ng/ML BMP4 and

20 ng/mL FGF2 for 5 days. The specified hepatic cells in RPMI/B27 supplemented with 20 ng/mL

HGF under 4% O2/5% CO2 were incubated for 5 days. For the final stage, hepatoblasts were

differentiated in HCM supplemented with 20 ng/mL OSM.

(B) Levels of HBsAg, HBV DNA and cccDNA in iPSC-derived hepatoblasts and iPSC-derived

hepatocytes. The results are shown as the mean ± SEM of 4 independent experiments. **p < 0.01.

45

Fig. 5. Isolation and characterization of fetal mouse LSECs and HSCs.

(A) Flow cytometric analysis of fetal mouse liver cells at E14.5.

(B) Primary culture of Stab2+ cells. Scale bar, 100 µm.

(C) Primary culture of Ngfr+ cells. Scale bar, 100 µm.

(D) Expression levels of LSEC markers in pre-sorting cells (pre-sort), Stab2+ cells (LSECs), and

Ngfr+ cells (HSCs). The results are shown as the mean ± SEM of 3 independent experiments. n =

3 in each group. ***p < 0.001.

(E) Expression levels of HSC markers in pre-sorting cells (pre-sort), Stab2+ cells (LSECs) and

Ngfr+ (HSCs) cells. The results are shown as the mean ± SEM of 3 independent experiments. ***p

< 0.001.

46

s, Carlsbad, CA, USA) supplemented with 10%

U ⁄ mL penicillin, 100 lg ⁄ mL streptomycin, and

non-essential amino acids (Life Technologies)

rwise described. Primary human hepatocytes

cells, isolated from urokinase-type plasminogen

nsgenic ⁄ SCID mice inoculated with PHH and

re purchased from PhoenixBio, Hiroshima, Japan

yoto, Japan respectively, and cultured under manprotocols. HepG2 ⁄ NTCP and HuH7 ⁄ NTCP cells

and HuH7-derived cell lines transduced by pCANand are susceptible to HBV infection.

We used a 1.2-fold HBV genome (isolate

enotype C, accession number AB246345) cloned

ndIII ⁄ EcoRI site of pUC19, termed pUC1.2x

e plasmid pUC1.2xHBV ⁄ NL was constructed by

nt (219–782) from the first codon of the HBV

ng frame and then inserting the NL gene (513 nt)

1 N1061 NLuc (Promega, Madison, WI, USA) by

n PCR cloning. Similarly, pUC1.2xHBV ⁄ NL+pol

cted by deleting 141 nt (295–436) from the first

e HBV preCore coding frame and then inserting

at the 178 nt position from the first codon of preby the In-Fusion method. The genome sizes of

d HBV ⁄ NL+pol were 3302 and 3731 nt, respecession of NL was designed to start from its own

pUC1.2xHBV-D, which produces all HBV proteins, has two

mutations in the encapsidation signal (CTGTGCC to

CTATGTC), and, thus, does not produce progeny virus. The

plasmid pUC1.2xHBV-D ⁄ MHD is mutated in the catalytic

domain, MDD, of HBV-D pol to MHD. The plasmid pCANNTCP-myc was constructed by inserting human NTCP cDNA

tagged with myc at the 30 -end into pCAN. The plasmid pX330

was obtained from Addgene (plasmid 42230; Cambridge, MA,

USA). Oligonucleotides designed for each target site were

inserted into the BbsI site of pX330.

Production of recombinant virus. The pUC1.2xHBV ⁄ NL,

pUC1.2xHBV ⁄ NL+pol, or pUC1.2xHBV ⁄ NL(-Met) plasmids

were cotransfected with pUCxHBV-D into HepG2 or HuH7

cells using Lipofectamine 3000 (Life Technologies). Medium

was harvested 3 or 4 days after transfection and the virus fraction prepared by precipitation with 13% PEG6000 (SigmaAldrich, St. Louis, MO, USA) containing 0.75 M NaCl. Virus

was further purified by precipitation through 20% sucrose in

TNE buffer (10 mM Tris [pH 7.6], 50 mM NaCl, 1 mM

EDTA) at 100 000 g for 3 h. Virus was then suspended in

Opti-MEM (Life Technologies) and stored at !80°C until use.

Infection and assay of NL activity. Cells were infected with

virus at a genome equivalent of 10–100 in the presence of 4%

PEG8000 and 2% DMSO overnight. Activity of NL and cell viability were then measured using the NL Luciferase Assay Kit

nomic structure of the reporter

uses (HBVs) and relative NanoLuc (NL)

ells infected by the viruses. (a)

of wild-type and reporter HBV, and

of the virus are shown. The indicated

of the pregenomes. A stretch of “A”s

ly A tail of the putative pregenomic

at rope with “E” indicates an

signal and “X” on that indicates

apsidation. The NL gene is inserted

ome so as to be translated from its

methionine. Virus was produced by

with

the

pUC1.2xHBV ⁄ NL,

NL+pol, or pUC1.2xHBV ⁄ NL(-Met) and

to HepG2 or HuH7 cells and the same

rus fractions was infected into PXB

cells were harvested 7 days

(A)after

Trans-well

d NL activity in cell lysates was

e NL activity in the cell lysates was

ean " SD; n = 3). The data shown are

endent experiments.

Fig. 6. Enhancement of HBV infection to iPSC-derived hepatocytes by fetal

mouse LSECs

co-culture system of iPSC-derived hepatocytes with fetal mouse NPCs.

(B) Schematic representation of the HBV-NL infection assay in iPSC-derived hepatocytes co-

ovember 2015 | vol. 106 | no. 11 | 1617

© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd

on behalf of Japanese Cancer Association.

cultured with fetal mouse NPCs.

(C) Relative HBV-NL activities in iPSC-derived hepatocytes (-), iPSC-derived hepatocytes co-

cultured with fetal mouse LSECs (LSEC), and fetal mouse HSCs (HSC). The results are shown as

the mean ± SEM of 3 independent experiments. **p < 0.01.

(D) Pregenomic structure of the reporter hepatitis B viruses (HBVs) and relative NanoLuc (NL)

47

activity in cells infected by the viruses. (a) Pregenomes of wild-type and reporter HBV/NL are

shown. The indicated sizes (kb) are of the pregenomes. A stretch of “A”s indicates a poly A tail of

the putative pregenomic RNA. A lariat rope with “E” indicates an encapsidation signal and “X” on

that indicates defect of encapsidation. The NL gene is inserted into the genome so as to be translated

from its own initiator methionine. (Nishitsuji et al., 2015)

48

Fig. 7. Enhancement of HBV infection to iPSC-derived hepatocytes by iPSCderived LSECs

(A) Trans-well co-culture system of iPSC-derived hepatocytes with iPSC-derived NPCs.

(B) Schematic representation of the HBV-NL and wild type HBV infection assay in iPSC-derived

hepatocytes co-cultured with iPSC-derived NPCs.

(C) Relative HBV-NL activities in iPSC-derived hepatocytes (-), iPSC-derived hepatocytes co-

cultured with iPSC-derived LSECs (iLSEC), and iPSC-derived HSCs (iHSC). The results are shown

as the mean ± SEM of 5 independent experiments. **p < 0.01.

(D) Levels of HBsAg, cccDNA and HBV DNA in iPSC-derived hepatocytes (-), iPSC-derived

hepatocytes co-cultured with iPSC-derived LSECs (iLSEC), and iPSC-derived HSCs (iHSC). The

results are shown as the mean ± SEM of 4 independent experiments. **p < 0.01.

49

Fig. 8. HBV infection to HepG2-NTCP cells co-cultured with iPSC-derived

LSECs in the trans-well system.

(A) Experimental design of the trans-well co-culture system of HepG2-NTCP cells with iPSC-

derived LSECs.

(B) Experimental design of HBV infection using HBV/NL and wild type HBV in the trans-well co-

culture system

(C) Relative HBV-NL activities in HepG2-NTCP cells (-) and HepG2-NTCP cells co-cultured with

iPSC-derived LSECs (iLSEC). The result is shown as the mean ± SEM of 3 independent

experiments. **p < 0.01.

(D) Levels of HBsAg, cccDNA and HBV DNA in HepG2-NTCP cells (-) and HepG2-NTCP cells

co-cultured with iPSC-derived LSECs (iLSEC). The results are shown as the mean ± SEM of 3

independent experiments. *p < 0.05.

50

Fig. 9. Detection of EGF in the supernatant of iPSC-derived LSECs by human

cytokine array analysis

iPS-derived LSECs were suspended in NPC maintenance medium and seeded at the density of

40,000 cells/cm2 in the upper chamber of trans-well. HepG2-NTCP maintenance medium was added

to the lower chamber and changed to fresh medium every day. The samples were prepared from the

lower chamber at day 5. Human cytokine antibody array membrane was used to detect cytokines

and the data were analyzed by ImageJ quantitatively. Trans-well inserts without iPSC-derived

LSECs was used as the control. Normalized mean pixel density = mean pixel density on sample

array x positive control on sample array/ positive control on control array - mean pixel density on

control array.

51

HBV infection in HepG2-NTCP

2.0

✱✱✱

Relative HBV DNA

1.5

anti-EGF

(2.5 µg/mL)

✱✱✱

1.0

ns

✱✱

0.5

ns

✱✱✱

✱✱✱

0.0

10

50

EGF (ng/mL)

Fig. 10. Modulation of HBV infection by EGF in HepG2-NTCP cells.

(A) Relative HBV DNA levels in HepG2-NTCP cells at 2 ng/mL, 10 ng/ml and 50 ng/ml of EGF

with or without 2.5 µg/mL anti-EGF antibody. The result is shown as the mean ± SEM of 3

independent experiments. **p < 0.01, ***p < 0.001.

(B) Southern blot analysis of HBV DNA fractions obtained from HepG2-NTCP cells infected with

HBV at post-infection day7.

52

relative HBV DNA

HBV internalization in HepG2-NTCP

2.0

1.5

***

1.0

***

**

**

**

10

25

50

100

0.5

0.0

anti-EGF

EGF (ng/mL)

Fig. 11. Modulation of HBV infection by EGF

Relative HBV DNA levels in HepG2-NTCP cells at 0 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/ml,

25 ng/mL, 50 ng/mL and 100 ng/ml of EGF in HBV internalization assay. The result is shown as

the mean ± SEM of 3 independent experiments. The result is shown as the mean ± SEM of 3

independent experiments. *p<0.05, **p < 0.01, ***p < 0.001.

53

Relative HBV DNA

HBV infection in primary human hepatocytes

2.5

**

2.0

1.5

1.0

**

**

***

0.5

0.0

10

25

50

100

EGF (ng/mL)

Fig. 12. Modulation of HBV infection by EGF in primary human hepatocytes.

Relative HBV DNA levels in primary human hepatocytes at 0 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL,

10 ng/ml, 25 ng/mL, 50 ng/mL and 100 ng/ml of EGF. The result is shown as the mean ± SEM of 3

samples in one experiment. *p<0.05, **p < 0.01, ***p < 0.001..

54

Relative HBsAg

HBV infection in iPS-hepatocytes

2.0

1.5

1.0

0.5

0.0

10

20

50

EGF (ng/mL)

Fig. 13. Modulation of HBV infection by EGF in iPS-hepatocytes.

Relative HBsAg level in iPS-hepatocytes at 0 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/ml, 20

ng/mL, and 50 ng/ml of EGF in HBV infection assay. The results of 5 independent experiments are

shown.

55

Fig. 14. Attachment of HBV by stimulation of EGF

(A, B) Relative HBV attachment in HepG2-NTCP cells treated with 2 ng/ml and 50 ng/ml of EGF

at 37˚C (A) and 4˚C (B). The results are shown as the mean ± SEM of 3 independent experiments.

*p < 0.05. **p < 0.01.

56

Dimer

Monomer

Fig. 15. EGFR expression on cell surface is modulated by EGF

Western blot analysis of cell surface protein treated with cross-linker BS3 at 0 ng/mL, 2 ng/mL and

50 ng/mL. monomer: 175 kDa, dimer: 350 kDa.

57

Fig. 16. Localization of EGFR and LAMP2

Immunofluorescence staining for EGFR (green) and LAMP2 (red) in HepG2-NTCP cells at 50

ng/ml of EGF at 37˚C and 4˚C. Scale bar, 10 µm. EGFR and LAMP2 are colocalized at 4˚C but

not at 37˚C.

58

Relative HBV DNA

HBV attachment with EGFR kinase inhibitor

1.6

control

1.4

gefitinib

✱✱

1.2

1.0

0.8

EGF (ng/mL)

Fig. 17. The extracellular HBV attachment is increased by the formation of active

dimer EGFR

Relative HBV attachment in HepG2-NTCP cells treated with or without 10 µM gefitinib and 2

ng/mL EGF. The results are shown as the mean ± SEM of 3 independent experiments. **p < 0.01.

Immunofluorescence staining for EGFR (green) and NTCP (red) in Hepg2-NTCP cells treated

with/without gefitinib at 2 ng/ml of EGF at 37˚C and 4˚C.

59

Relative HBV DNA

HBV attachment in EGFR-KD cells

1.6

si-control

1.4

si-EGFR

1.2

EGFR

GAPDH

✱✱✱

tro

FR

sisi-

1.0

0.8

EGF (ng/mL)

Fig. 18. Knockdown of EGFR expression cancelled the enhanced HBV attachment

by EGF in HepG2-NTCP

Relative HBV attachment in EGFR knock down HepG2-NTCP cells treated with or without 2

ng/mL EGF. The results are shown as the mean ± SEM of 3 independent experiments. *p < 0.05.

N.S., not significant.

60

Fig. 19. Knockdown of EGFR expression cancelled the enhanced HBV attachment

by EGF in HepG2-NTCP and HepG2 cells

Relative HBV attachment in EGFR knock down HepG2-NTCP and HepG2 cells treated with or

without 2 ng/mL EGF. The results are shown as the mean ± SEM of 3 independent experiments.

*p < 0.05. N.S., not significant.

61

Fig. 20. HBV is internalized via the TK-independent CME pathways without EGF

stimulation

(A) Relative HBV internalization in EGFR knockdown HepG2-NTCP cells pretreated with 2 ng/ml

of EGF. The results are shown as the mean ± SEM of 3 independent experiments. *p < 0.05. **p

< 0.01. ***p < 0.001. N.S., not significant.

(B) Relative HBV infection levels in HepG2-NTCP cells (-), HepG2-NTCP cells treated with 10

µM gefitinib. The results are shown as the mean ± SEM of 3 independent experiments. *p < 0.05.

**p < 0.01. ***p < 0.001. N.S., not significant.

62

Fig. 21. Gefitinib inhibits EGFR endocytosis in the presence of EGF

Immunofluorescence staining for EGFR (green) and NTCP (red) in HepG2-NTCP cells treated with

or without 2 ng/ml of EGF and 10 µM gefitinib.

63

HBV infection with CME inhibitor

Relative HBV DNA

2.0

Control

IKA

1.5

✱✱✱

1.0

✱✱✱

ns

0.5

0.0

50

EGF (ng/mL)

Fig. 22. HBV is internalized via the CME pathways at low dose of EGF

(A) Relative HBV infection levels in HepG2-NTCP cells (-), HepG2-NTCP cells treated with

ikarugamycin (IKA). The results are shown as the mean ± SEM of 3 independent experiments. *p

< 0.05. **p < 0.01. ***p < 0.001. N.S., not significant.

64

Fig. 23. IKA inhibits EGFR internalization at a low dose of EGF

Immunofluorescence staining for EGFR (green) and NTCP (red) in HepG2-NTCP cells at 2 ng/ml

of EGF treated with or without 2 µM IKA. Scale bar, 10 µm.

65

HBV infection with CIE inhibitor

Relative HBV DNA

2.0

Control

FLP

1.5

1.0

✱✱✱

0.5

0.0

50

EGF (ng/mL)

Fig. 24. HBV is internalized via the CIE pathway at a high dose of EGF

Relative HBV infection levels in HepG2-NTCP cells (-), HepG2-NTCP cells treated with filipin

(FLP). The results are shown as the mean ± SEM of 3 independent experiments. *p < 0.05. **p <

0.01. ***p < 0.001. N.S., not significant.

66

Fig. 25. FLP inhibits EGFR internalization at a high dose of EGF

Immunofluorescence staining for EGFR (green) and LAMP2 (red) in HepG2-NTCP cells at 2 ng/ml

of EGF treated with or without 1 µM FLP. Scale bar, 10 µm.

67

HBV infection with lysosome inhibitor

Relative HBV DNA

2.0

Control

CQ

1.5

ns

1.0

ns

✱✱✱

0.5

0.0

50

EGF (ng/mL)

Fig. 26. HBV is degraded in the lysosome at a high dose of EGF

Relative HBV levels in HepG2-NTCP cells (-), HepG2-NTCP cells treated with chloroquine (CQ).

The results are shown as the mean ± SEM of 3 independent experiments. *p < 0.05. **p < 0.01.

***p < 0.001. N.S., not significant.

68

Fig. 27. CQ inhibits EGFR degradation at a high dose of EGF

Immunofluorescence staining for EGFR (green) and LAMP2 (red) in HepG2-NTCP cells at 2 ng/ml

and 50 ng/mL of EGF treated with 25 µM CQ. Scale bar, 10 µm.

69

Fig. 28. NTCP and EGFR are not colocalized after internalization

Immunofluorescence staining for EGFR (green) and NTCP (red) in HepG2-NTCP cells treated with

PreS1-TAMRA (white) and HBV at 2 ng/ml of EGF treated. Scale bar, 10 µm.

70

Fig. 29. Competition between HBV and PreS1 in cells with or without EGF

stimulation

Relative HBV attachment (A), relative HBV infection (B) in HepG2-NTCP cells treated with or

without 1µM PreS1 and 50 ng/mL EGF. Inhibition rate = (1- PreS1/control) x100%. The results

are shown as the mean ± SEM of 3 independent experiments. **p < 0.01. ***p < 0.001.

71

Fig. 30. Summary

72

Table 1. List of quantitative PCR primers for mouse genes

gene

Left Primer

Right Primer

Stab2

TGTCCAGACGGCTACATCAA

CCAGGGATATCCAGGACGTA

Lyve1

CCTCCAGCCAAAAGTTCAAA

TCCAACACGGGGTAAAATGT

Pecam1

CTGGTGCTCTATGCAAGCCT

AGTTGCTGCCCATTCATCAC

F8

TCATGTATAGCCTGGATGGGA

GATGAGTCCACATTGCCAAA

Ngfr

GTGTGCGAGGACACTGAGC

GGGGGTAGACCTTGTGATCC

Desmin

GTGAAGATGGCCTTGGATGT

CTCGGAAGTTGAGAGCAGAGA

Hgf

CCTGACACCACTTGGGAGTA

CTTCTCCTTGGCCTTGAATG

Actb

TTCTTTGCAGCTCCTTCGTT

ATGGAGGGGAATACAGCCC

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Table 2. List of quantitative PCR primers and probes for HBV detection

Primer/Probe

HBSF2

CTTCATCCTGCTGCTATGCCT

HBSR2

AAAGCCCAGGATGATGGGAT

cccDNA F7

TCCCCGTCTGTGCCTTCTC

cccDNA R7

GCACAGCTTGGAGGCTTGA

cccDNA P7

FAM- CCGTGTGCACTTCG

74

Table 3. List of primary and secondary antibodies used for

immunocytochemistry analysis and FCM analysis of mouse and human cells

Primary Antibody

Supplier

LAMP2

abcam (ab25631)

EGFR

cell signaling (D38B1)

Myc

abcam (ab206486)

Stabilin2

Nonaka et al.

LNGFR

miltenyibiotec (REA648)

Secondary Antibody

Supplier

Alexa Fluor 488 anti-Rabbit IgG

Invitrogen (A32790)

Alexa Fluor 555 anti-Mouse IgG

Invitrogen (A21424)

Alexa Fluor 647 anti-Mouse IgG

Invitrogen (A28181)

75

...