ORIGINAL RESEARCH
Transmission of HBV DNA Mediated by Ceramide-Triggered
Extracellular Vesicles
Takahiro Sanada,1,* Yuichi Hirata,1,* Yutaka Naito,2 Naoki Yamamoto,1 Yoshiaki Kikkawa,3Yuji Ishida,4 Chihiro Yamasaki,4 Chise Tateno,4 Takahiro Ochiya,2 and Michinori Kohara1
1

Department of Microbiology and Cell Biology, 3Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science,
Setagaya-ku, Tokyo, Japan; 2Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku,
Tokyo, Japan; 4PhoenixBio Co, Ltd, Higashi-Hiroshima, Hiroshima, JapanSUMMARY
Extracellular vesicle is a nanovesicle that shuttles proteins,
nucleic acids, and lipids, thereby inﬂuencing cell behavior. This
article shows that ceramide-triggered extracellular vesicles
work as DNA cargo for hepatitis B virus-DNA and are capableof trasmitting to naive hepatocytes. Further, this article
demonstrates that the transmission of hepatitis B virus-DNA
via these extracellular vesicles is resistant to antibody
neutralization.
BACKGROUND & AIMS: An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby inﬂuencing cell behavior. A recent crop of reports have shown that EVs are
involved in infectious biology, inﬂuencing host immunity and playing
a role in the viral life cycle. In the present work, we investigated the
EV-mediated transmission of hepatitis B virus (HBV) infection.
METHODS: We investigated the EV-mediated transmission of
HBV infection by using a HBV infectious culture system thatuses primary human hepatocytes derived from humanizedchimeric mice (PXB-cells). Puriﬁed EVs were isolated byultracentrifugation. To analyze the EVs and virions, we usedstimulated emission depletion microscopy.
RESULTS: Puriﬁed EVs from HBV-infected PXB-cells were shownto contain HBV DNA and to be capable of transmitting HBV DNA
to naive PXB-cells. These HBV-DNA–transmitting EVs wereshown to be generated through a ceramide-triggered EV
production pathway. Furthermore, we showed that these HBVDNA–transmitting EVs were resistant to antibody neutralization;
stimulated emission depletion microscopy showed that EVs lacked
hepatitis B surface antigen, the target of neutralizing antibodies.
CONCLUSIONS: These ﬁndings suggest that EVs harbor a DNA
cargo capable of transmitting viral DNA into hepatocytes during
HBV infection, representing an additional antibodyneutralization–resistant route of HBV infection. (Cell Mol Gastroenterol Hepatol 2017;3:272–283; http://dx.doi.org/10.1016/
j.jcmgh.2016.10.003)
Keywords: HBV; Extracellular Vesicles; Transmission Pathway.

DNA.1,2 Although an effective HBV preventive vaccine isavailable, HBV infection continues to be a major healthconcern.3 Approximately 350 million people are chronicallyinfected by HBV worldwide, and some of these infections will
result in cirrhosis and hepatocellular carcinoma.2,4There is growing evidence that HBV maturation andegress depend on intraluminal vesicles of maturing endosomes known as multivesicular bodies (MVBs).5–7 HBV coreand L envelope proteins have been shown to interact withthe ubiquitin-interacting adaptor g2-adaptin and the Nedd4
ubiquitin ligase, possibly to regulate transport of the viralstructures through the late endosomal pathway.8,9 In
addition, several studies have shown that Vps4A/B andAlix/AIP1 (a functional link between endosomal sorting
complexes required for transport (ESCRT)-I and ESCRT-III
in mammalian cells) contribute to HBV release.5,6 Thus,
HBV virions are thought to bud into late endosomes or
MVBs by using ESCRT/Vps4B functions, subsequently exiting the cell by the extracellular vesicle (EV) pathway.
EVs fall into 2 general categories based on biogenetic
deﬁnition.10 One category generally is referred to as theexosome, which consists of 30- to 100-nm diameter nanovesicles that originate from the intraluminal vesicles of
MVBs; the fusion of MVBs with the cell membrane results in
the release of vesicles into the extracellular compartment.11
The other category generally is referred to as microvesicles.
Microvesicles generally are larger than exosomes (40- to
1000-nm diameter) and bud from the cellular membrane.10
Historically, EVs were thought to function primarily in the*Authors share co-ﬁrst authorship.
Abbreviations used in this paper: anti-HBs, antibody to hepatitis B
surface antigen; BSA, bovine serum albumin; ESCRT, endosomal
sorting complexes required for transport; EV, extracellular vesicle;
GEq, genome equivalent; HA, hemagglutinin; HBc, hepatitis B core;
HBcAg, hepatitis B core antigen; HBIG, hepatitis B immune globulin;
HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; mRNA,
messenger RNA; MVB, multivesicular body; PXB-cells, primary humanhepatocytes derived from chimeric mice with human liver; nSMase,
neutral sphingomyelinase; nts, nucleotides; PBS, phosphate-bufferedsaline; qPCR, quantitative real-time polymerase chain reaction; STED,
stimulated emission depletion.
Most current article© 2017 The Authors. Published by Elsevier Inc. on behalf of the AGA

H

epatitis B virus (HBV) is an infectious agent thatbelongs to the Hepadnaviridae family, whose members are characterized by a partially double-stranded circular

Institute. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2352-345X
http://dx.doi.org/10.1016/j.jcmgh.2016.10.003

March 2017

removal of unnecessary proteins from the cell. More recent
studies have shown that EVs are involved in multiple biological processes; under normal and pathologic conditions,EVs appear to shuttle their contents (including proteins,
lipids, and nucleic acids) among various cells.12–14
Notably, recent studies have shown that EVs play animportant role in infectious disease by mediating thetransfer of pathogen-derived antigens and virulence factors.15 EVs derived from virus-infected cells contain viralproteins and viral RNA; the contents of these vesicles are
enclosed in a cellular membrane, thus enabling viruses toevade host immune response to herpes simplex virus,16,17
human immunodeﬁciency virus,18,19 Epstein–Barr virus,17
and cytomegalovirus20 infection. Furthermore, EVs fromhepatitis A virus or hepatitis C virus–infected hepatocytes
permit the respective viruses to invade and replicate within
host hepatocytes.21,22 However, the role of EVs in HBV
infection still largely is unknown. Speciﬁcally, the lack ofviral culture systems for HBV has hampered the evaluationof EV-mediated transmission of HBV infection.
In this study, we investigated EV-mediated transmissionof HBV infection in a highly efﬁcient HBV infectious culture
system, for which we used primary hepatocytes derived
from humanized chimeric mice with human livers. We
further elucidated the involvement of ceramide-triggered EV
production in HBV morphogenesis.

Transmission of HBV DNA by EVs

273

HBV-DNA Quantiﬁcation
DNA extraction from samples was performed using
SMItest EX-R&D kits (Nippon Genetics, Tokyo, Japan)
according to the manufacturer’s instructions.
Quantiﬁcation of HBV genomic DNA was performed byquantitative polymerase chain reaction (qPCR) using theThunderbird Probe qPCR Mix (Toyobo, Osaka, Japan) CFX96
real-time PCR detection system (Bio-Rad, Hercules, CA).
Ampliﬁcation reaction mixtures (30 mL) contained 5 mL DNA
solution, 200 nmol/L forward primer HB-166-S21 (nucleotides [nts] 166–186; 5’-CACATCAGGATTCCTAGGACC-3’),
200 nmol/L reverse primer HB-344-R20 (nts 344–325;
5’-AGGTTGGTGAGTGATTGGAG-3’), 300 nmol/L TaqMan(Thermo Fisher Scientiﬁc, Waltham, MA) probe HB-242-S26FT(nts 242–267; 5’-CAGAGTCTAGACTCGTGGTGGACTTC-3’), and
15 mL of Thunderbird Probe qPCR Mix. Thermal cycling conditions were as follows: activation of uracil-N-glycosylase(UNG) by incubation at 50 C for 2 minutes; activation of Taqpolymerase (Nippongene, Toyama, Japan) and inactivation of
UNG by incubation at 95 C for 10 minutes; and ampliﬁcation
by 53 cycles of incubation at 95 C for 20 seconds and 60 C for
1 minutes. The standard curve for this quantiﬁcation was
calculated using a series of 10-fold dilutions (at deﬁned concentrations) of a recombinant plasmid into which the HBVgenome had been inserted.

Indirect Immunoﬂuorescence Analysis

Materials and Methods
Ethics Statement
This study was performed in strict accordance with boththe Guidelines for Animal Experimentation of the Japanese
Association for Laboratory Animal Science and the recommendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. All protocols wereapproved by the ethics committee of the Tokyo Metropolitan
Institute of Medical Science (license number 15059).

Primary Hepatocytes From Humanized
Chimeric Mice: PXB-Cells
Primary human hepatocytes derived from chimeric micewith human liver (PXB-cells; PhoenixBio, Hiroshima, Japan)
were cultured on type I, collagen-coated, 48-well plates
using maintenance medium as previously described.23
Culture ﬂuids or cells from wells with the same treatment
in a plate were pooled, and then used for analysis.

Inocula Used for Infection of Primary
Hepatocytes
Inocula of HBV genotype A or C were derived fromserum obtained from humanized chimeric mouse at 8–10
weeks after infection by the respective virus.

Infection of PXB-Cells by HBVPXB-cells were inoculated with HBV at 5 genome
equivalents (GEq) per cell. Inoculated cells were washed at
1 and 2 days after infection, and culture medium wascollected every 5 days thereafter.

Cells were ﬁxed with 10% phosphate-buffered formalin(pH 7.4) for 10 minutes at room temperature. After washing 3
times with phosphate-buffered saline (PBS; pH 7.4), cells werepermeabilized with 0.25% Triton X-100 (Nacalai Tesque,
Kyoto, Japan) for 10 minutes at room temperature. Viral antigen was stained with antibody to hepatitis B surface antigen(anti-HBs) rabbit serum (1:300) and Alexa-488–conjugatedanti-rabbit IgG (Thermo Fisher Scientiﬁc). Nuclei also were
stained using 4’,6-diamidino-2- phenylindole.

In Situ Hybridization
PXB-cells were ﬁxed in 4% buffered paraformaldehyde(pH 7.5) for 1 hour at room temperature. After washing 3
times with PBS, the cells were treated with 0.25% Triton
X-100 for 10 minutes at room temperature. To detect HBVDNA, in situ hybridization was performed as described
previously using fragmented RNA probes synthesized froma full-length HBV genome.24

EV Isolation
The culture ﬂuids of PXB-cells were centrifuged at 2000  g
for 10 minutes at 4 C. To remove further cellular debris, thesupernatants were centrifuged at 12,000  g for 30 minutes at
4 C, and then ﬁltered through a 0.22-mm ﬁlter. The resulting
ﬁltered culture supernatant (designated as Sup in experiments) then was processed for isolation of EVs as follows. Sup
was ultracentrifuged at 110,000  g for 70 minutes at 4 C. The
resulting supernatants were decanted and retained separately
for subsequent use (see later) as the fraction without EVs (EV-).
Meanwhile, the ultracentrifugation pellets were resuspended
in PBS and subjected to a second round of ultracentrifugation

274

Sanada et al(110,000 g for 70 min at 4 C). The resulting supernatants
were discarded, and the pellets were resuspended in PBS,
yielding the EV fraction (EVþ) used for subsequent analyses.

RNA Extraction and Analysis
Total RNA was extracted from the PXB-cells and from theliver of humanized chimeric mouse using the RNeasy Mini Kit(Qiagen, Hilden, Germany). Expression of messenger RNAs(mRNAs) in PXB-cells was determined by an Agilent Human
Gene Expression 4  44k v2 microarray (Agilent Technologies,
Santa Clara, CA). Expression of mRNAs in the liver of humanized chimeric mouse was determined by mRNA sequencing(Illumina HiSeq; Illumina, San Diego, CA). The expression ofneutral sphingomyelinase (nSMase) 1, 2, and 3 were determined by TaqMan Gene Expression Assays (nSMase1,
Hs00162006_m1; nSMase2, Hs00920354_m1; and nSMase3,
Hs00215775_m1; Thermo Fisher Scientiﬁc).
Total RNA was extracted from the culture ﬂuid and EV
fractions using SepaGene RV-R (Eidia, Tokyo, Japan), andreverse-transcribed using the High-Capacity Complementary
DNA Reverse Transcription Kit (Thermo Fisher Scientiﬁc)according to the manufacturer’s protocol. Viral complementary DNA then was quantiﬁed by qPCR as described earlier.

Immunoprecipitation
Each aliquot (200 mL at 3.0  104 viral DNA copies/mL) of
Sup, EV-, and EVþ fractions from primary human hepatocytes
was mixed with an equal volume of PBS containing 1% bovine
serum albumin (BSA) and 5 mg of one of the following: anti-CD9
antibody (M-L13; Becton, Dickinson and Company, Franklin
Lakes, NJ), anti-CD63 antibody (MEM-259; Abcam, Cambridge,UK), anti-CD81 antibody (JS-81; Becton, Dickinson and Company), or anti-hemagglutinin (HA) tag antibody (12CA5(2);BioVision, San Francisco, CA), or with diluted serum (1:300)
from a normal (HBV-naive) or HBs-immunized mouse. The
resulting mixture was incubated for 16 hours at 4 C. Eachﬂuid þ antibody mixture then was combined with 50 mL of a
50% slurry of protein G–Sepharose 4 Fast Flow (GE Healthcare, Chicago, IL) and incubated with rotation at 4 C for 2
hours. The mixtures were centrifuged at 2300  g for 20
seconds at 4 C, and the supernatants were discarded. The
pellets were washed twice with 1% BSA in 10% Dulbecco’s
modiﬁed Eagle medium. The pellets then were resuspended in
10% Dulbecco’s modiﬁed Eagle medium containing 1% BSA
and used for quantiﬁcation analysis of HBV DNA.

Treatment With GW4869
PXB-cells were infected with HBV. At 22 days postinfection, the cells were incubated with culture medium
containing GW4869 (Merck Millipore, Billerica, MA) at theindicated concentrations. After 4 days’ incubation, the culture ﬂuid and cells were collected, pooled, and used foranalysis of the HBV-DNA titer.

Inoculation of HBV From Each Fraction
to PXB-Cells
The Sup was collected from the HBV-infected PXB-cells.
The EVþ and the EV- fractions were prepared as described

Cellular and Molecular Gastroenterology and Hepatology Vol. 3, No. 2earlier. PXB-cells were inoculated with 5 GEq/cell of HBVderived from the Sup, EVþ, or EV- fractions.

Neutralization Test
For time-course analysis, a volume of the supernatantcontaining 2  106 HBV-DNA copies was incubated with 1
IU hepatitis B immune globulin (HBIG; Mitsubishi Tanabe
Pharma Corporation, Osaka, Japan) in 500 mL of culture
medium for 1 hour at room temperature. Naive PXB-cells
were cultured with the HBIG-pretreated culture supernatantfor 2 or 24 hours. After the incubation period, the cells werecollected, pooled, and used for analysis.
For neutralization analysis of Sup, EV-, and EVþ fractions, HBV-vaccinated human serum or AB human serum(Pel-Freez Biologicals, Rogers, AR) diluted 1:10 in culture
medium was mixed with an equal volume of Sup, EVþ, orEV- containing 2.0  106 viral DNA copies. These mixtures
were incubated at 37 C for 1 hour, and then 250 mL of each
mixture was used to inoculate a PXB-cell culture (4.0  105
cells, 5 GEq/cell). For analysis of viral entry, at 3 hours after
infection, the cells were washed 5 times with PBS and thencollected for analysis. For long-term analysis, the cells were
washed at 1 and 2 days postinfection and the culture ﬂuid
was harvested every 5 days thereafter. Collected culture
ﬂuids were pooled and used for qPCR analysis.

Scanning Electron Microscopy
Samples for scanning electron microscopy were prepared by spotting of microvesicles (suspended in 0.1% PBS)
onto poly-L-lysine–coated (Sigma-Aldrich, St. Louis, MO)coverslips. The samples were ﬁxed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer ﬁxative solution for 30
minutes at room temperature and then washed 3 times with
0.1 mol/L phosphate buffer solution. The samples were
immersed in a 1% (wt/vol) OsO4 solution for 1 hour at 4 C,
then dehydrated in a graded ethanol series and transferred
to t-butyl alcohol. The samples were dried in a freeze dryer(Hitachi ES-2020; Hitachi High-Tech Fielding Corporation,
Tokyo, Japan), coated with OsO4 for 1 minute using an
osmium plasma coater (NL-OPC80; Nippon Laser and Electronics Laboratory, Aichi, Japan), and examined using a
Hitachi S-4800 ﬁeld emission scanning electron microscopeat an accelerating voltage of 10 kV.

Stimulated Emission Depletion Microscopy
Samples of the culture ﬂuid or EVs were ﬁxed with 4%paraformaldehyde on poly-L-lysine–coated coverslips for 30
minutes at room temperature. The samples were washedtwice with PBS; incubated with 50 mmol/L NH4Cl for 10
minutes at room temperature; washed twice with PBS;
permeabilized with 50 mg/mL digitonin for 10 minutes atroom temperature; washed twice with PBS; treated with
0.25% Triton X-100 for 10 minutes at room temperature;
washed twice with PBS; and blocked with blocking buffer(1% BSA and 1 mmol/L EDTA in PBS) at 4 C overnight. The
slides then were incubated with primary antibodies for 2
hours at room temperature. The primary antibodies wereused at the following concentrations or dilutions: anti-CD81

March 2017

antibody and biotin-conjugated anti-CD81 antibody, 2 mg/
mL; anti–hepatitis B core antigen (anti-HBc) mouse serum,
1:100; anti-HBs rabbit serum, 1:600. The slides were
washed 5 times with PBS containing 0.05% Tween 20 andthen incubated with 40 mg/mL secondary antibody (Alexa
488– or Alexa 555–conjugated anti-mouse, anti-rabbit, or
streptavidin; Thermo Fisher Scientiﬁc) for 2 hours at room
temperature. After washing, the specimens were mounted
using Prolong Diamond (Thermo Fisher Scientiﬁc) andexamined using a TCS SP8 stimulated emission depletion(STED) 3 microscope (Leica Microsystems, Wetzlar,
Germany).

DNase I Treatment
The DNase I reaction mixture consisted of 26 mL of Sup,
EV-, or EVþ fractions (or extracted DNA as a control), 1 mL
of DNase I (10 U/mL; GE Healthcare), and 3 mL of DNase I
buffer. The mixture was incubated at 37 C for 10 minutes.
The reaction mixture then was incubated at 75 C for 10
minutes to heat-inactivate the DNase I. DNA extraction fromsamples was performed using SMItest EX-R&D kits (Nippon
Genetics) according to the manufacturer’s instructions, andviral DNA was quantiﬁed by qPCR as described earlier.

Statistical Analysis
Statistical analyses were performed with Prism software(version 6.0; GraphPad, GraphPad Software, La Jolla, CA).
Statistical signiﬁcance was determined by using 2-tailed
1-way analysis of variance with a post hoc Bonferroni
or Mann–Whitney U test. P values less than .05 wereconsidered signiﬁcant.

Results
Primary Hepatocytes Derived From Humanized
Chimeric Mice Support HBV Propagation
Several in vitro studies have reported that HBV can
infect the primary hepatocytes derived from human andtree shrew, or any of several cell lines.25 Here, to evaluate
EV-mediated HBV transmission, we used primary hepatocytes derived from humanized chimeric mice with humanlivers (PXB-cells).23 First, we examined whether PXB-cells
supported HBV propagation. As indicated in the schematic
protocol (Figure 1A), supernatant was collected for 42 days
after infection with HBV; both genotypes A and C were
tested (separately). HBV DNA in the resulting culture
supernatants was measured by real-time qPCR. Both genotypes of HBV propagated well in PXB-cells and the viral DNA
level in culture ﬂuid reached approximately 107–108
copies/mL (on day 42) (Figure 1B). Furthermore, immunoﬂuorescent analysis and in situ hybridization analysis
showed that HBV infected approximately 75% of the
PXB-cells on day 41 (Figure 1C).
Next, we tested if supernatant from HBV-infected
PXB-cell culture showed infectivity for naive PXB-cells.
The time-course analysis of the supernatants obtained fromthe supernatant-infected cells showed that HBV-DNA levels
increased to approximately 107–108 copies/mL on day 37

Transmission of HBV DNA by EVs

275(Figure 1D). These results indicated that HBV infected andreplicated in PXB-cells, and that the supernatant of HBVinfected PXB-cells showed the same infectivity of naive
PXB-cells as the serum of HBV-infected humanized chimericmice. We next used this system to examine EV-mediated
HBV transmission.

EVs Isolated From HBV-Infected Primary
Hepatocytes Contain HBV DNA
Recent studies have shown that EVs from hepatitis C
virus–, cytomegalovirus-, and Epstein–Barr virus–infected
cells contain RNA and proteins for the respective
viruses.17 However, it still is not known whether EVs canharbor a viral DNA cargo. To test this possibility, we
assessed whether EVs from the culture supernatant of
HBV-infected PXB-cells contain HBV DNA. qPCR analysisof the EVþ fraction isolated from HBV-infected PXB-cells
showed that 62.5% ± 39.8% of HBV DNA in the culture
supernatant was carried in the EV fraction (Figure 2A).
The concentrations of HBV RNA in the Sup and in theEV- fraction were 0.021 ± 0.015 and 0.018 ± 0.016 of
those of HBV DNA in each fraction, respectively, whereas
the corresponding ratio in the EVþ fraction was 0.0018 ±0.0004 (Figure 2B). We next immunoprecipitated the Supwith antibodies (anti-CD9, anti-CD63, and anti-CD81)
against representative exosome membrane proteins andthen determined HBV-DNA levels in the immuneprecipitation samples. The HBV-DNA levels of specimen
recovered from immune-precipitation with anti-CD9,
CD63, and CD81 antibodies were 0.84 ± 0.18, 2.08 ±
1.77, and 2.17 ± 0.64, respectively, of that recovered from
immune-precipitation with control antibody (Figure 2C).
These observations are consistent with microarray andRNA sequencing data (Figure 2D and E), indicating that
CD81 (but not CD9) is highly expressed in HBV-infected
PXB-cells and livers. Next, to analyze what fraction of
HBV virions (Dane particles) and EVs carrying HBV DNA
are contained in the 3 fractions (Sup, EV-, and EVþ), we
immune-precipitated the 3 fractions with antibodies
against HBs and CD81, and then determined HBV-DNAlevels in the immune-precipitation samples. In specimens recovered from immune-precipitation with anti-HBs
antibody, specimens derived from EV- were enriched for
HBV DNA, whereas HBV DNA was not detected in specimens derived from EVþ (Figure 2F). In contrast, specimens recovered from immune-precipitation of EVþ with
anti-CD81 antibody were enriched for HBV DNA
compared with those derived by anti-CD81 immuneprecipitation of Sup or EV- (Figure 2F). Considered
together, the results of this experimental series suggested
that EVs derived from HBV-infected PXB-cells carry viral
DNA as a cargo, but that these particles apparently are
depleted for HBV RNA.

GW4869 Treatment Suppresses the Productionof HBV-DNA–Containing EVs
Next, we investigated the mechanisms that generate HBVDNA–containing EVs. Two major pathways have been

Sanada et al

Cellular and Molecular Gastroenterology and Hepatology Vol. 3, No. 2

A
Time

B
Cell
seeding

HBV infection(Genotype A, C)(Day)
Day -1

Day 0

MediumSampling
Every 5 days

HBV-DNA (copies/mL)

276

HBV-DNA in supernatant

1.0E+09
1.0E+08
1.0E+07
1.0E+06

Genotype A
Genotype C

1.0E+05
1.0E+04
0

10

20

30

40

50

Days after HBV infection

C

0 dpi

41 dpiD
HBV-DNA in supernatant

0 dpi

41 dpi

HBV-DNA (copies/mL)

1.0E+08

1.0E+07

1.0E+06

1.0E+05
0

10

20

30

40

50

Days after HBV infection

Figure 1. Primary hepatocytes derived from humanized chimeric mice with human livers support HBV propagation. (A)Experimental schedule of HBV infection of PXB-cells. (B) Time-course studies of the PXB-cells inoculated with chimeric mouseserum samples positive for HBV genotypes A or C. HBV-DNA levels in supernatants were determined by qPCR (n ¼ 3–4 pergroup at each time point). (C) Detection of viral DNA by in situ hybridization (upper) and of HBs antigen by immunoﬂuorescentassay (lower) from HBV-infected PXB-cells at 41 days postinfection (dpi). Upper: Viral DNA, blue. Lower: HBs antigen, green;
nuclei, blue. (D) Time-course studies of the PXB-cells inoculated with supernatants from HBV-infected PXB-cells (n ¼ 3). (Band D) Data were pooled from 3 or 4 independent experiments with 1 plate per experiment. (C) The data were reproducible in 3
independent experiments with 1 plate per experiment, and the ﬁgure shown here is a representative one. (B and D) Values arepresented as means ± SDs.

implicated in exosome generation: one is an ESCRT-mediatedprocess,11,26 and the other is a ceramide-triggered process.27
In other work with HBV-infected humanized chimeric mice
(Figure 3A and B), we observed that serum and liver HBVDNA levels correlated with accumulation of the transcript
encoding nSMase2, a key enzyme for ceramide-mediatedexosome generation. On the basis of those results, we hypothesized that the production of HBV DNA from HBVinfected hepatocytes depends on the ceramide-mediatedexosome pathway. To test this prediction, we treated HBVinfected PXB-cells with GW4869, an nSMase inhibitor thatis known to inhibit ceramide biosynthesis.27,28 Treatment
with GW4869 attenuated HBV-DNA levels in culture supernatants, but not in hepatocytes (Figure 3C and D). These results suggested that inhibition of ceramide biosynthesis
suppresses the production of HBV-DNA–containing EVs. In
conﬁrmation of this hypothesis, we observed that GW4869
treatment suppressed the number of EVs visible in electron

microscopic images of puriﬁed EVs isolated from HBVinfected PXB-cells (Figure 3E and F). In addition, GW4869
treatment resulted in decreased HBV-DNA levels in the puriﬁed EV fraction when normalized per milliliter of the original culture supernatant, consistent with the decrease of thenumber of EVs (Figure 3G and H). Considered together, theseresults indicated that HBV-DNA–containing EVs are produced
via the ceramide-triggered exosome pathway.

STED Microscopic Analysis Shows That EVs
Incorporate CD81 and HBcAg Protein
To clarify whether or not the HBV-DNA–containing EVs
possess viral proteins, we examined the interaction ofhepatitis B surface antigen (HBsAg) and hepatitis B core
antigen (HBcAg) with EVs labeled with anti-CD81 antibody.
The interaction was examined using STED microscopy,
which permits the detection of co-located EVs and viralMarch 2017

Transmission of HBV DNA by EVs

A

B

1.2

10
Copies(Normalized to DNA)

Viral DNA copies(Normalized to Sup)

1.0
0.8
0.6
0.4
0.2
Sup

RNA

EV-

EV+

1
0.1
0.01

EV+

Sup

CD9

CD63

2

P = .131
0

4Viral DNA copies(Normalized to Ctrl Ab)

3

CD81

P = .14

4
Viral DNA copies(Normalized to Ctrl Ab)

4
Viral DNA copies(Normalized to Ctrl Ab)

DNA

0.001

0

C

3
2
1
0

Control Ab Anti-CD9(Anti-HA-Tag)

Control Ab Anti-CD63(Anti-HA-Tag)

D PXB-cells

E Liver

70,000250

P = .020
P = .062
P = .00078

P = .0008

32
1
0

Control Ab Anti-CD81(Anti-HA-Tag)

P = .0015
P = .0000084
P = .0000013

60,000200

40,000FPKMCounts

50,00030,000

150
100

20,00050

10,000
0

CD9

CD63

0

CD81

CD9

F

P = .43
P = .011
P = .052
400

P = .043

Sup

P = .006

EV-

300

P = .26

EV+
200

100

s
i-H
nt
A

m
al
m
or
N

B

se
ou

81
D
i-C
nt
A

C
e
yp
ot
Is

o

an
t

ib

on

od

tr
o

y

l

0

N

HBV-DNA (copies) from 6.0 × 103 copies
input of each fraction (Sup, EV-, and EV+)

Figure 2. Characterization of extracellular vesicles derived from HBV-infected
hepatocytes. (A) The ratio of viral DNA in
EV fractions to viral DNA in Sup (n ¼ 3). (B)The ratio of HBV-DNA:HBV-RNA copies inSup, the EV- fraction, and the EVþ fraction(n ¼ 3). (C) HBV-DNA levels (determined by
qPCR) after immune-precipitation of fractions with each antibody. Values arenormalized to HBV DNA of control antibody(Control Ab) in the respective experiment(n ¼ 3). (D) Expression of mRNAs encoding
CD9, CD63, or CD81 in PXB-cells as
determined by an Agilent Human Gene
Expression 4  44k v2 microarray. (E)Expression of mRNAs encoding CD9,
CD63, or CD81 in liver from humanizedchimeric mouse as determined by mRNA
sequencing (Illumina HiSeq). (F) HBV-DNAlevels (determined by qPCR) in Sup, EVfraction, and EVþ fraction after immuneprecipitation of each fraction containing
6.0  103 viral DNA copies with each antibody (n ¼ 3). Data were pooled from (A–D,and F) 3 independent experiments with 1
plate per experiment, or (E) from 3 individual
animals. Values are shown as means ±SDs. Ctrl, control; HA-Tag, hemagglutinin
tag.

277

CD63

CD81

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Cellular and Molecular Gastroenterology and Hepatology Vol. 3, No. 2

A

nSMase1 r = 0.064
nSMase2 r = 0.75
nSMase3 r = 0.07

3
2.5
2
1.5
1
0.5
0
1.0E+10

2.5
2
1.5
1
0.5
0

1.5E+105.0E+06 1.0E+07 1.5E+07 2.0E+07
HBV-DNA in liver (copies/ug of DNA)HBV-DNA in serum (copies/mL)

N.S.

C

N.S.

D

P = .019

HBV-DNA (copies/μg DNA)

3.0E+072.0E+07

1.0E+07

0

1

N.S.

N.S.

5.0E+07
4.0E+073.0E+072.0E+07
1.0E+070

10 (μM)

3

N.S.
6.0E+07

4.0E+07
HBV-DNA (copies/mL)

30
5.0E+09

00

nSMase1 r = 0.23
nSMase2 r = 0.58
nSMase3 r = 0.23

3.5
Relative expression to
HBV (-) chimeric mice liver

Relative expression to
HBV (-) chimeric mice liver

3.5

B

0

1

GW486910 (μM)

3

GW4869

E
H

G
15

10

5

0

-

P = .03

1.5E+07
HBV-DNA (copies/mL)F

EV number (number/fields)

20

P = .0024+

GW4869proteins. Imaging analysis with STED microscopy showedthat CD81 co-localized with HBcAg but not with HBsAg(Figure 4A and B). Furthermore, the frequency of HBcAgþ
CD81þ spots was 25% that of CD81þ spots, a value comparable with that of HBcAgþ HBsAgþ spots (Figure 4E).
These results suggested that HBV-DNA–containing EVs
possess HBcAg, but not HBsAg. This result motivated us to1.0E+07

5.0E+06

0-+
GW4869Figure 3. GW4869 treatment suppresses thesecretion of HBV-DNA–
containing EVs. (A and B)
The correlation betweennSMase 1, 2, and 3 and (A)serum or (B) liver HBV
DNA in HBV-infected humanized chimeric mice(n ¼ 7). Each symbol represents a distinct animal.
To measure the strength of
the association, the Pearson correlation coefﬁcient
was calculated. (C and D)
HBV-DNA levels (determined by qPCR) in (C)culture supernatant and(D)
PXB-cells
after
GW4869 treatment (n ¼ 3).
(E and F) Electron microscopic images of puriﬁed
EV fraction isolated from
HBV-infected
PXB-cells
treated with (E) vehicle or(F) GW4869. White bar,
100 nm. (G) The number of
puriﬁed EV and (H) copy
number of HBV DNA in EV
fraction isolated from
HBV-infected
PXB-cellswith (þ) or without (-)
GW4869 treatment (10mmol/L) (n ¼ 3). Data werepooled from (A and B) 7
individual animals, (C, D,
and H) 3 independent experiments with 1 plate perexperiment, or from (G)
3–4 ﬁelds per experiment,or (E and F) data wererepresentative of 3 independent experiments with
1 plate per experiment.
Values are shown as (C, D,
and H) means ± SDs or (G)SEM.

examine the effect of anti-HBs neutralizing antibody on HBV
entry and replication. We performed a time-course analysis
of PXB-cells inoculated with HBV that had been pretreatedwith HBIG (Figure 4F). Neutralizing antibodies decreased
intracellular HBV-DNA level by 70% compared with HBVDNA levels in nontreated hepatocytes 2 hours after infection. Furthermore, in the presence of neutralizing

March 2017

Transmission of HBV DNA by EVs

A

B

F

P = .00067
P = .00011

P = .42500
HBV-DNA (copies/μg DNA)

6.0E+05
5.0E+05
Number/mm2

D

C

E

279

4.0E+05
3.0E+05
2.0E+04
1.0E+04

400
300
200
100
0

0
CD81+

CD81+HBc+

HBs+

HBs+HBc+

HBIG-

HBIG+

2 h after infection

HBIG-

HBIG+

24 h after infection

Figure 4. Characterization of HBV-containing infectious EVs. (A–D) STED microscopic images of supernatant of HBVinfected PXB-cells. Scale bars: 2.5 mm. (A) Green, CD81; red, HBcAg; arrowhead, CD81þHBcAgþ spot; inset, enlarged image of CD81þHBcAgþ spot; (B) green, CD81; red, HBsAg; (C) green, HBcAg; red, HBsAg; arrowhead, HBcAgþHBsAgþ spot;
inset, enlarged image of HBcAgþHBsAgþ spot; and (D) isotype control. (E) Number of CD81þ, CD81þHBcAgþ, HBsAgþ, andHBcAgþHBsAgþ spots observed from 20  20 mm ﬁelds. Values are shown as means ± SD for values normalized per mm2 of
3 independent determinations. (F) Intracellular HBV-DNA levels of primary human hepatocytes at 2 or 24 hours after HBV
infection (n ¼ 3). HBIG- and HBIGþ indicate that the cells were inoculated without or with (respectively) HBIG pretreatment.
(A–D) The data were reproducible in 3 independent experiments with 1 plate per experiment, and the ﬁgures shown here arerepresentative images from one such experiment. Data were pooled from (E) 3 independent experiments with 1 ﬁeld per
experiment, or (F) 1 plate per experiment. Values are shown as means ± SDs.

antibodies, HBV-DNA levels in nontreated hepatocytes at
24 hours after infection increased 3-fold compared with
HBV-DNA levels at 2 hours after infection, and increased
1.6-fold even in the HBIG-treated hepatocytes. These results
suggested that HBV DNA is transmitted and replicates evenin the presence of neutralizing antibodies.

EV Fractions Transmit HBV DNA to Primary
Hepatocytes via a Mechanism That Is Resistantto the Effects of Neutralizing Antibodies
Next, we focused on the transmission of HBV mediatedby EVs. Speciﬁcally, we isolated puriﬁed EVs from the culture supernatant of HBV-infected PXB-cells, and then used
the EVþ fraction to infect naive PXB-cells; this procedure isshown schematically in Figure 5A. qPCR analysis conﬁrmedthat this procedure yielded detectable levels of HBV DNA at
3 hours after infection by the EVþ fraction (Figure 5B). We
noted, however, that HBV-DNA levels still were approximately 10-fold lower after infection with EVþ comparedwith levels obtained after infection with Sup or EV- fractions. We inferred that the higher HBV levels observed upon
infection with the Sup and EV- fractions reﬂected thepresence of HBV virions in the Sup and EV- fractions,and that these virions were removed from the EVþ fraction
via ultracentrifugation. To clarify the effect of ultracentrifugation on HBV virions and HBV-DNA–containing
EVs, we examined the number of CD81þHBcAgþ and
HBsAgþHBcAgþ particles using STED microscopy before
and after ultracentrifugation. As shown in Figure 5C, thenumber of HBcAgþHBsAgþ spots showed a larger depletion
than that of the HBcAgþCD81þ spots. That is, the number of280

Sanada et al

Cellular and Molecular Gastroenterology and Hepatology Vol. 3, No. 2

A

B
1.0E+04
HBV-DNA (copies/μg DNA)

Ultracentrifugation

Supernatant(EV-)

PXB-cells with
HBV infection

Supernatant(Sup)

Pellet(EV+)

Mix andincubate (1h)Sup, EV-, or EV+

Incubate (3h)P = .00073

P = .0017

P = .57
1.0E+03NT
1.0E+02

Ctrl AbnAb

1.0E+01

Measurement of
HBV-DNANaïve PXB-cells

1.0E+00

Sup

EV-

EV+

Ctrl Ab or nAb

C

D
2.0E+06

N.S.

*

HBs+HBc+

1.0E+07
HBV-DNA (copies/ml)

Number (spots/mL)

1.6E+06

1.2E+06
P = .039

8.0E+05*

1.0E+08

CD81+HBc+

4.0E+05

1.0E+06**

1.0E+05Sup
EVEV+

1.0E+04
1.0E+03

0

-

2

+

7

12

17

22

Days postinfection

UltracentrifugationE
HBV-DNA copies(Normalized to samples without DNase I treatment)*

F
EV/HBV core particle

Dane particle

1.6
1.4
P = .013

1.2

Anti-HBs Ab

1
0.8

?

0.6

?

HBs Ag0.4
0.2

Transcription

N.D.

0

-

+
Sup

-

+
EV-

-

+
EV+

-

+

?

DNase I

Naked DNA(Control)HBc Ag

cccDNA

Reversetranscription

RNA packaging

Figure 5. HBV-DNA–containing EVs show infectivity. (A) Depiction of the infection experiment procedure. (B) HBV-DNAlevels in cells at 3 hours postinfection with the indicated HBV fraction. As indicated, each fraction was either not pretreated(NT) or pretreated with control antibody (Ctrl Ab) or with neutralizing antibody (nAb) (n ¼ 3). (C) Numbers of HBcAgþCD81þ
and HBcAgþHBsAgþ spots before (-) and after (þ) ultracentrifugation by STED microscopic images (n ¼ 3). (D) HBV-DNAlevels in culture ﬂuid at each time point after infection with the indicated HBV fraction (n ¼ 3). *HBV-DNA level of cells
infected with the EVþ fraction was signiﬁcantly higher than those of cells infected with the Sup or EV- fraction. (E) Ratio of
HBV-DNA levels in the respective samples treated with and without DNase I (n ¼ 3). Samples consisted of Sup, EV-, or EVþ
fractions, or of naked (control) DNA. N.D., not detected. (F) Proposed mechanisms of HBV propagation and secretion. In allcases, data were pooled from 3 independent experiments with 1 plate per experiment, and values are shown as means ± SDs.

HBcAgþCD81þ spots was 2.6-fold that of HBcAgþHBsAgþ
spots after ultracentrifugation, suggesting an enrichment for
HBV-containing EVs compared with HBV virions. Next, toshow that infection by the EVþ fraction reﬂected an
EV-speciﬁc role, we investigated whether the transmissionof HBV was inhibited by the presence of neutralizing
antibodies—that is, whether pre-incubation with virus(virion)-speciﬁc antibodies interfered with the infection
process. For virus entry analysis, the cells were collected at
3 hours postinfection and the viral DNA in cells wasanalyzed. As shown in Figure 5B, HBV-DNA levels after

infection with either Sup or EV- were inhibited signiﬁcantlyin the presence of neutralizing antibodies. In contrast, theuse of neutralizing antibodies did not decrease HBV-DNAlevels after infection with the EVþ fraction. In addition,
we analyzed the replication ability of HBV-containing EVs.
PXB-cells were infected with Sup, EV-, or EVþ to determine
whether HBV DNA transmitted by EVs supported viral
replication in the transmitted hepatocytes. Culture ﬂuid
from HBV-infected PXB-cells was collected for 22 days
postinfection, and the titer of viral DNA was quantiﬁed. The
viral DNA titer from EVþ-infected PXB-cells increased

March 2017

progressively over the course of the experiment; a similar
pattern was observed after infection with the other fractions(Figure 5D). These results showed that the HBV DNA
derived from EV was replicative. Finally, to test whether
viral DNA is encapsidated or just attached to the membraneof the EVs, we analyzed the DNase I resistance of the HBVDNA carried by the EVþ fraction. As Figure 5E shows,control DNA (naked DNA) subjected to DNase I treatment
was not detected by qPCR. In contrast, approximately 40%
of HBV DNA in the EVþ fraction was resistant to DNase I
treatment. This result indicated that at least 40% of HBVDNA was encapsidated in EVs.
The results of this experimental series indicated that EVs
can transmit HBV infection even in the presence ofneutralizing antibodies, that is, via a non–virion-mediatedprocess (Figure 5E).

Discussion
EVs are presumed to function primarily in intercellularcommunication. These vesicles can inﬂuence the immune
system; act as signaling complexes; transfer receptors fromone cell to another; and convey speciﬁc mRNAs, microRNAs,
and proteins between cells, thereby impacting numerous
physiological processes.12–14 EVs also facilitate viral transport, spread cell damage, and stimulate malignant transformation.17 However, the role of EVs in HBV infection stilllargely is unknown. The lack of a viral culture system
for HBV has hampered the evaluation of EV-mediatedtransmission of HBV infection.
Several HBV culture systems have been used in previous
in vitro HBV analyses.25 Primary human hepatocytes andprimary tree shrew hepatocytes are susceptible to HBV
infection, but the limited availability of, and genetic variation among, such primary cells makes such culture systems
difﬁcult to use.29 Recent work has shown that sodium
taurocholate cotransporting polypeptide–overexpressing
cell lines show susceptibility to HBV infection and support
HBV propagation.30 However, the efﬁciency of HBV infection
and production in this cell line is reduced compared withthat in primary hepatocytes. In other work, Ishida et al23
reported that PXB-cells, which are primary hepatocytes
from chimeric mice, show high susceptibility to HBV infection and support persistent infection and viral production athigh titers (approximately 106 copies/mL in the supernatant). Furthermore, these cells are more readily available,
and show less genetic variation, than primary human or tree
shrew hepatocytes. Because PXB-cells are primary hepatocytes, the characteristics of these cells are assumed to better
resemble those of human hepatocytes compared with hepatoma cell lines. Taking these advantages into consideration,
we propose that the PXB-cell culture system currently is thebest model for analysis of the clinical effects of HBV.
By using this HBV infectious culture system, we showedthat the EV fraction derived from HBV-infected PXB-cells
contains abundant HBV DNA (representing 60% of all HBVDNA in the culture supernatant), but is relatively depleted
for HBV RNA. We further showed that HBV-DNA–containing
EVs are capable of HBV transmission to naive PXB-cells.

Transmission of HBV DNA by EVs

281

Several previous studies have reported that viral RNA istransmitted via EVs,21,22 but this report shows that EVs
carry DNA as cargo and are capable of transmitting viral
DNA into hepatocytes.
Two major pathways have been implicated in exosome
generation: one is an ESCRT-mediated process,11,26 and theother is a ceramide-triggered process.27 HBV core and L
envelope proteins have been shown to interact with theubiquitin-interacting adaptor, g2-adaptin, and the ubiquitin
ligase, Nedd4, in a process thought to regulate transport ofviral structures through an ESCRT-mediated process.8,9 In
contrast, a role for ceramide in HBV transmission has notbeen reported. In the present study, we showed that
GW4869, an inhibitor of ceramide-triggered EVs, suppresses
HBV DNA in the cell supernatant but not in cells. Furthermore, GW4869 treatment decreases EV production. The
decrease of EV number in the supernatant was comparablewith the decrease in the level of HBV DNA in the EV fraction(when values were normalized per milliliter of supernatant), suggesting that GW4869 suppresses the production of
HBV-DNA–containing EVs. Thus, HBV-DNA–containing EVs
appear to be generated through the ceramide-triggered
pathway, although the role of the ESCRT pathway in the
production of HBV-containing infectious EVs remains to beelucidated. The combination of the present and previous
work shows that HBV exploits both sorting pathways,
with ESCRT-mediated processes used in the generation ofDane particles and ceramide-triggered processes in thegeneration of HBV-DNA–transmitting EVs.
We have shown here that the transmission of HBV DNA
to naive PXB-cells occurs via EV-enclosed HBV DNA, andthat this transmission is resistant to the effects of neutralizing antibodies. This observation is consistent with the lackof HBsAg in HBV-DNA–transmitting EVs, as conﬁrmed by
STED microscopic analysis. It has been reported that 10%–30% of newborns from HBsAg-/HBeAg-positive mothers
cannot be protected by passive/active vaccination alone andbecome chronic HBV carriers.3 It also has been reported thatasymptomatic occult HBV infections are frequent even in
individuals who have protective levels of anti-HBs antibodies.3 These reports implied that some virus evades
antibody neutralization. Our ﬁndings suggest that HBVcontaining infectious EVs may contribute to transmission
even in the presence of antibody neutralization, separate
from the effect of HBV S gene escape mutants.3
We showed that even in the presence of neutralizing antibody, 30% of the HBV inoculum still was capable of infecting
naive PXB-cells (Figure 4F), a process thought to be mediatedby HBV-DNA–transmitting EVs. Our DNase I treatment
experiment showed that although the viral DNA of Sup and EVwere not degraded by the DNase I treatment, approximately
60% of the viral DNA of EVþ was degraded and 40% of theviral DNA was resistant (Figure 5E). This result was consistent
with the low efﬁciency of the attachment of EVþ to PXB-cells(Figure 5B). Given that the HBV EVs in the Sup fraction werecompletely resistant to DNase I treatment, we postulate thatthe HBV EVs in the EVþ fraction may be damaged by the ultracentrifugation step, and that this damage may affect the
efﬁciency of EV attachment to the hepatocytes.

282

Sanada et al

After the inoculation of PXB-cells with EVþ, the viral DNA
level of the culture ﬂuid showed a gradual increase. Thisobservation suggested that HBV DNA transmitted by EVs
replicates in hepatocytes. Interestingly, although the EVþ
fraction showed a low efﬁciency of attachment to hepatocytes, the viral DNA level in the culture ﬂuid of PXB-cells
infected with EVþ was signiﬁcantly higher than the viral DNA
level in the culture ﬂuids of cells inoculated with Sup orEV-. EV is known to contain various host factors (eg, microRNAs), and these factors may enhance the initial replicationof HBV. Indeed, some microRNAs are reported to enhance thereplication of HBV.31,32 To clarify this point, further studies
focusing on HBV-DNA replication of EV will be needed.
In summary, we have shown, using primary hepatocytes
derived from humanized chimeric mouse, that HBV DNAcontaining EVs are generated through ceramide-triggered
vesicle formation, and that these EVs are infectious to primary hepatocytes. Furthermore, HBV transmission by theseEVs is resistant to antibody neutralization because thesevesicles lack HBs antigen, as shown by STED microscopic
analysis. These results suggest the existence of a separate
pathway for transmission of HBV; distinct strategies may beneeded to address HBV infection by this pathway.

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Received October 19, 2015. Accepted October 14, 2016.
Correspondence
Address correspondence to: Michinori Kohara, PhD, Department ofMicrobiology and Cell Biology, Tokyo Metropolitan Institute of Medical
Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. e-mail:kohara-mc@igakuken.or.jp; fax: (81) 3-5316-3137.
Acknowledgments
The authors are very grateful to Ms Rie Hayashi from Leica Microsystems fortechnical support.
Current address of Y.H.: Columbia University Medical Center, New York,New York.
Conﬂicts of interest
These authors disclose the following: Yuji Ishida, Chihiro Yamasaki, and Chise
Tateno are employees of PhoenixBio Co, Ltd. The remaining authors disclose
no conﬂicts.
Funding
This work was supported by grants from the Ministry of Health Science andWelfare of Japan (H24-HBV-general-014) and the Research Program on
Hepatitis from the Japanese Agency for Medical Research and Development(15fk0310015h0004). The funders had no role in the study design, datacollection and analysis, decision to publish, or preparation of the manuscript.