Download Mouse Hepatic Cells Support Assembly of Infectious

GASTROENTEROLOGY 2011;141:1057–1066
BASIC AND TRANSLATIONAL—LIVER
Mouse Hepatic Cells Support Assembly of Infectious Hepatitis C Virus
Particles
GANG LONG, MARIE–SOPHIE HIET, MARC P. WINDISCH, JI–YOUNG LEE, VOLKER LOHMANN, and
RALF BARTENSCHLAGER
See editorial on page 806.
BACKGROUND & AIMS: Hepatitis C virus (HCV) has a
high propensity to establish persistence; better understanding of this process requires the development of a
fully permissive and immunocompetent small animal
model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin
to allow entry of HCV. However, RNA replication is poor
in mouse cells, and it is not clear whether they support
assembly and release of infectious HCV particles. We used
a trans-complementation-based system to demonstrate
HCV assembly competence of mouse liver cell lines.
METHODS: A panel of 3 mouse hepatoma cell lines that
contain a stable subgenomic HCV replicon was used for
ectopic expression of the HCV structural proteins, p7,
nonstructural protein 2, and/or apolipoprotein E (apoE).
Assembly and release of infectious HCV particles was
determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. RESULTS: Mouse replicon cells released low amounts of
HCV particles, but ectopic expression of apoE increased
release of infectious HCV to levels observed in the human
hepatoma cell line Huh7.5. Thus, apoE is the limiting
factor for assembly of HCV in mouse hepatoma cells but
probably not in primary mouse hepatocytes. Products of
all 3 human alleles of apoE and mouse apoE support HCV
assembly with comparable efficiency. Mouse and human
cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. CONCLUSIONS: Mouse hepatic cells
permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse
model of HCV infection.
Keywords: HCV Mouse Model; HCV Assembly; Liver Disease; Virology.
H
epatitis C virus (HCV) is a leading cause of acute
and chronic liver diseases, affecting 130 –170 million people worldwide.1 Up to 80% of infected individuals
are unable to clear the virus and have a high risk of
developing cirrhosis and hepatocellular carcinoma.2 There
is no HCV-specific vaccine available, and the only approved therapy, pegylated interferon-␣ combined with
ribavirin, has numerous adverse effects and limited efficacy.3
HCV has a positive-strand RNA genome encoding a
polyprotein that is cleaved co- and post-translationally
into 10 products.4 Core protein and envelope glycoprotein
1 (E1) and E2 are main constituents of the virus particle,
whereas p7 and nonstructural protein 2 (NS2) are auxiliary assembly factors. NS3, NS4A, NS4B, NS5A, and
NS5B constitute the viral replicase that catalyzes the amplification of the viral genome.
HCV assembly and release is tightly linked to the very
low density lipoprotein (VLDL) biosynthesis pathway.5,6
Inhibition of microsomal triglyceride transfer protein or
knockdown of apolipoprotein B (apoB) was reported to
reduce production of infectious HCV.7,8 Additionally,
apoE is a constituent of infectious HCV particles,9 and
depletion reduces infectivity titers.10 –12
Although the establishment of a fully permissive culture system has greatly advanced molecular analyses of
the viral replication cycle,13–15 there is still an unmet need
for an HCV permissive immunocompetent small animal
model. A major obstacle to its development is the very
narrow host range of the virus that infects only humans
and chimpanzees. Although entry of HCV into mouse
cells can be achieved by ectopic expression of the human
entry factors CD81 and occludin16 or by using an HCV
mutant adapted to the use of murine CD81,17 RNA replication is extremely limited.18,19 Only when using a selection approach and subgenomic replicons, mouse hepatoma cell lines such as Hepa1-6, MMH1-1, or AML12
containing stably replicating HCV RNAs can be obtained.20,21 However, given the lack of stable replication of
Abbreviations used in this paper: apoB, apolipoprotein B; apoE, apolipoprotein E; GFP, green fluorescent protein; DMEM, Dulbecco’s modified minimal essential medium; (h), human; HA, hemagglutinin; HCV,
hepatitis C virus; HCVTCP, trans-complemented HCV particles; IRES,
internal ribosome entry site; LDL, low-density lipoprotein; LDLR, LDL
receptor; (m), mouse; NS, nonstructural protein; SCARB1, scavenger
receptor class B type I; TCID, tissue culture infective dose 50; TCP,
transcomplementation particle; VLDL, very low density lipoprotein.
© 2011 by the AGA Institute
0016-5085/$36.00
doi:10.1053/j.gastro.2011.06.010
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Department of Infectious Diseases, Molecular Virology, Medical Facility, Heidelberg University, Heidelberg, Germany
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complete HCV genomes in mouse liver cells, it is unknown whether they support assembly and release of
infectious HCV particles.
Taking advantage of mouse liver cell lines containing a
self-replicating HCV replicon and a trans-complementation approach, we demonstrate that mouse hepatic cells
are permissive for assembly and release of infectious HCV
particles. A key factor is the expression level of apoE.
Importantly, human and mouse apoE are equally efficient
in supporting HCV particle production, arguing against a
species restriction of the late steps of the HCV replication
cycle in mouse cells.
Materials and Methods
Cell Culture and Reagents
The cell lines Huh7.5 and Hep56.1D (CLS-Cell Line
Service)22 were cultured in Dulbecco’s modified minimal essential medium (DMEM; Invitrogen, Carlsbad, CA) supplemented
with 2 mmol/L L-glutamine, nonessential amino acids, 100 U
penicillin per milliliter, 100 ␮g streptomycin per milliliter, and
10% fetal calf serum (complete DMEM). Huh7.5 and Hep56.1Dderived cell lines containing a subgenomic replicon and the
trans-complementing expression cassette were kept in complete
DMEM containing 100 ␮g/mL of G418 (to select for the replicon) and 5 ␮g/mL of blasticidin (to select for expression of core
to NS2). AML12-sgJFH1 and MMH1-1-sgJFH1 cells were cultured as previously described.20
Plasmid Construction
The plasmids pFK-Jc123 and pSGR-JFH1-H2476L24 have
been described recently. Construction of further plasmids is
described in Supplementary Materials and Methods.
Electroporation and Selection of Stable
Replicon Cells
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In vitro transcripts of individual constructs were generated as previously described.25 For electroporation of HCV RNA
into cell lines Huh7.5 and Hep56.1D, trypsinized cells were
washed twice with phosphate-buffered saline and used for transfection and selection as described in Supplementary Materials
and Methods.
Characterization of HCV Particles by
Iodixanol Density Gradient Analysis and
Neutralization Assays
Concentrated trans-complemented HCV particles
(HCVTCP) were analyzed by density gradient centrifugation as
described in the Supplementary Materials and Methods. For
neutralization assays, Huh7.5 cells were inoculated at a multiplicity of infection of 0.01 in the presence or absence of a given
antibody. Two hours later, cells were washed twice with phosphate-buffered saline and cultured in DMEM complete for 72
hours. Cells were fixed and analyzed by immunohistochemistry
as described in the Supplementary Materials and Methods.
Virus Purification by Using Flag- or
Hemagglutinin-Specific Affinity Gel
These methods are described in the Supplementary Materials and Methods.
GASTROENTEROLOGY Vol. 141, No. 3
Results
Transient and Stable Replication of Genomic
HCV RNAs in Mouse Cells
The primary goal of our study was to determine
HCV assembly competence of mouse cells for which we
selected the cell line Hep56.1D that has excellent growth
and transfection properties (not shown). In the initial set
of experiments, we transiently transfected Hep56.1D cells
with the HCV genome Jc1 and quantified virus production. Although considerable amounts of core protein were
produced, no infectivity was detected (data not shown).
Given the very low replication of HCV in mouse
cells,20,21 which might have limited virus production, we
attempted to establish mouse cell-derived clones containing selectable Jc1 genomes. Although cell clones could be
obtained, no infectivity was detected because of accumulation of deletions affecting the region encoding the structural proteins (not shown).
Production of Infectious HCV Particles in
Mouse Liver Cells
To overcome these technical limitations, we devised an alternative strategy based on trans-complementation of subgenomic HCV replicons with the viral
“assembly factors” (core E1, E2, p7, and NS225). A JFH-1derived selectable subgenomic replicon containing the cell
culture-adaptive mutation H2476L in NS5B24 was transfected into Hep56.1D cells (Figure 1A). Upon selection
with G418, cell clones could be obtained containing HCV
RNA of the correct size (Figure 1B). The cell clone with
highest HCV RNA amounts (clone 3; designated Hep56sgJFH-cl3) was transduced with a lentiviral expression
construct encoding for the assembly factors (Figure 1A).
Western blot analysis of this cell clone designated Hep56sgJFHcl3-CNS2 confirmed expression of the assembly factors and the presence of the JFH1 replicon (Figure 1C).
Culture supernatants did not contain a statistically significant higher amount of HCV RNA released from cells
expressing the assembly proteins as compared with the
replicon cells (Figure 1D). However, when using the more
discriminating tissue culture infective dose 50 (TCID50)
assay to monitor infectivity titers, a detectable amount of
infectious particles was found in supernatants of Hep56sgJFHcl3-CNS2 cells but not with replicon control cells
(Figure 1E). This result suggested production of infectious HCV particles in Hep56.1D mouse liver cells, albeit
with low efficiency. Given the origin of these particles, by
analogy to our earlier report,25 they were designated muHCVTCP to indicate the use of a trans-complementation
(TCP) approach in mouse (mu) cells.
Expression of Human or Mouse apoE in
Mouse Cells Promotes Production of Infectious
muHCVTCP Particles to the Same Extent
In a search for the reasons of this low efficiency, we
argued that host factors playing a crucial role in HCV
particle production might either be incompatible between
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Figure 1. Production of infectious HCV particles in mouse cells by using trans-complementation. (A) A schematic representation of the selectable
subgenomic JFH1 replicon neo-sgJFH1-H2476L is shown in the top. The neo gene is fused at the 5= end with the 16 5= terminal codons of the core
gene. The respective fusion protein is expressed via the HCV IRES residing in the 5= NTR. The replicase genes (NS3 to NS5B) are translated via the
IRES of the encephalomyocarditis virus (EI). H2476L refers to the position of the cell culture-adaptive mutation in NS5B. The trans-complementing
assembly cassette (core to NS2) expressed via a lentiviral vector is drawn below. The coding region is derived from the Jc1 genome and composed
of the core to p7 region of the HCV isolate J6; the NS2 gene is chimeric and composed of the first transmembrane domain of the J6 isolate, whereas
the remainder of NS2 (indicated by the dashed line) is derived from the JFH-1 isolate.23 (B) Detection of replicon RNA in Hep56.1D cell clones by
Northern blot analysis. For each cell clone, 5 ␮g total RNA was analyzed. Left 2 lanes: Given amounts of in vitro transcripts spiked with total RNA from
naïve cells served as size marker and to estimate HCV RNA amounts. Total RNA from naïve Hep56.1D (control cells) was used as negative control.
␤-actin (lower panel) was used as loading control. Positions of replicon RNA (HCV) and 28S ribosomal RNA are indicated in the left. (C) Western blot
analysis of Hep56.1D cells and cell lines derived thereof 3 weeks after lentiviral transduction, for proteins specified on the right of each panel. Positions
of molecular size markers are indicated on the left. For comparison of apoE amounts contained in 105 Hep56.1D cells, total protein in a homogenate
of given numbers of mouse liver cells was analyzed in parallel. Actin was used as loading control. (D) Intra- and extracellular HCV RNA amounts in
replicon cells with or without stable expression of core to NS2. Total RNA prepared 48 hours after seeding from cells or culture supernatant of 105
cells was quantified by using quantitative reverse-transcription polymerase chain reaction. (E) Detection of infectivity in culture supernatants of cells
specified in the bottom by using a limiting dilution assay. Data in panels D and E represent the mean of 3 independent assays; error bars represent
standard deviations from the means.
mouse and human or limiting in Hepa56.1D cells. To
address the latter possibility, a comparative transcriptome analysis between naïve Hep56.1D cells and 3 independent replicon-containing cell clones was conducted. Most striking was the low expression of apoE in
Hep56.1D cells (Figure 1C) that was reduced even further in the 3 replicon cell clones (Supplementary Figure
1). In contrast, in a homogenate prepared from the
same number of mouse liver cells, apoE was highly
abundant, arguing that low apoE expression is a property of Hep56.1D cells. We therefore concluded that
apoE, which is essential for HCV particle production,
might limit assembly in Hep56.1D cells but probably
not in mouse liver cells.
Focusing our analysis on apoE, we studied the impact
of different apoE expression levels and possible species
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Figure 2. HCV particle production is limited in mouse cells by (m)apoE amounts. (A) Schematic representation of constructs used for expression of
authentic or tagged apoE genes, respectively. (B) Western blot analysis of Hep56-sgJFHcl3-CNS2 cells transiently transfected with constructs given
in the top Cells were transfected with equal amounts of the expression construct, and, 48 hours later, proteins were analyzed. Molecular sizes are
indicated on the left of each blot, and proteins are specified on the right. Actin served as loading control. (C–F) Hep56-sgJFHcl3-CNS2 cells were
transfected with constructs specified in the bottom of each panel; 48 hours later, cells and supernatants were harvested and used for determination
of (C) released core protein by enzyme-linked immunosorbent assay, (D) released HCV RNA by quantitative reverse-transcription polymerase chain
reaction, and (E) intra- and (F) extracellular infectivity by using limiting dilution assay. Mock-transfected cells and cells transfected with the
pcDNA-GFP expression construct served as negative controls. Dashed lines in C and D indicate assay backgrounds. Shown are the means of 3
independent experiments; error bars indicate standard deviations from the means.
restriction. An amino acid sequence alignment between
mouse (m) apoE, which is the only allele in this species,
and the predominant human (h) apoE3 gene revealed a
similarity of 80.9% (Supplementary Figure 2) and an
8-amino acid deletion in (m)apoE. We therefore constructed apoE expression vectors, with or without a C-terminal hemagglutinin (HA)-affinity tag (Figure 2A), the
latter allowing the unambiguous detection of apoE proteins and their quantification.
Transient transfection of Hep56-sgJFHcl3-CNS2 cells
each with equal amounts of an apoE construct demonstrated comparable expression level of all HA-tagged proteins, thus allowing direct comparisons in assembly assays
(Figure 2B). Importantly, (h)apoE3-HA and (m)apoE-HA
shared a similar subcellular localization pattern (Supplementary Figure 3), suggesting that the fusion with the HA tag
did not affect apoE properties. Finally, core and NS5A amounts
were not affected by apoE overexpression (Figure 2B).
We next studied the impact of apoE expression on HCV
particle production in mouse cells. Hep56-sgJFHcl3CNS2 cells were transfected with the various apoE constructs or the green fluorescent protein (GFP) control or
mock-transfected and release of core protein and viral
RNA as well as intra- and extracellular infectivity were
assessed (Figure 2C–F). Expression of each individual
apoE led to a slight increase of core protein release as
compared with the control cells independent from the
apoE species origin (Figure 2C). In contrast, when we
measured RNA amounts in the culture supernatants, we
did not detect a difference (Figure 2D). Instead, we found
that high amounts of RNA were released from replicon
cells independent from the structural proteins (Supplementary Figure 4), which is similar to observations made
with replicons in human hepatoma cells.26 However, with
the more discriminatory TCID50 assay, we observed an
unambiguous difference between Hep56-sgJFHcl3-CNS2
control cells (GFP and mock) and cells transfected with
any of the apoE expression constructs (Figure 2E and F).
In fact, in all cases of apoE expression, extracellular infectivity titers were enhanced about 100-fold as compared
with Hep56-sgJFHcl3-CNS2 expressing only limiting
amounts of endogenous (m)apoE. Most importantly, (h)apoE and (m)apoE enhanced production of muHCVTCP
particles to the same extent, arguing that apoE is not a
determinant of HCV species specificity. We also did not
detect a difference between the various (h)apoE alleles,
showing that they all support HCV assembly to the same
extent.
Finally, the comparable infectivity titers attained with
authentic and HA-tagged apoE demonstrates full functionality of the latter. We note that production of infectious muHCVTCP particles was also achieved in 2 other
Hep56.1D-derived replicon cell clones (numbers 8 and 10;
Figure 1B), thus excluding a cell clone-specific effect (data
not shown). We also note that Hep56.1D replicon cells
expressing very low amounts of apoE do not release (noninfectious) HCV particles (Supplementary Figure 5), arguing that the high release of viral RNA and core from such
cells is either nonspecific or reflects the release of naked
noninfectious particles or RNA-containing replication
complexes.26,27 In conclusion, our results demonstrate
that Hep56.1D mouse cells are permissive for HCV assembly and that expression of (m)apoE is a limiting factor.
Biophysical Properties of Mouse Cell-Derived
Infectious muHCVTCP Particles
To study biophysical properties of mouse cell-derived particles and the impact of apoE on association with
lipids and lipoproteins, muHCVTCP particles released from
Hep56-sgJFHcl3-CNS2 cells after transfection with GFP
control or apoE constructs were analyzed by equilibrium
density gradient centrifugation (Figure 3). HCV RNA, core
protein, and infectivity in each fraction were quantified.
Cells transfected with the GFP vector released substantial
amounts of viral RNA that sedimented to very low density
in agreement with our earlier report on nonspecific RNA
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release from replicon cells (Figure 3A).26 In contrast, cells
transfected with either of the apoE expression construct
released RNA-containing structures with peak density of
about 1.1 g/mL similar to the one of HCV particles produced in Huh7 cells.5 A high amount of core protein was
detected in the supernatant of the Hep56-sgJFHcl3-CNS2
cells transfected with the GFP control plasmid and these
core-containing structures had a high density (⬃1.18
g/mL), whereas the peak of Core protein released from
apoE-transfected cells was much broader and covered the
range from 1.07 to 1.18 g/mL (Figure 3B). Most importantly, culture supernatants of apoE-transfected cells contained high amounts of infectivity with a peak density of
⬃1.1 g/mL similar to HCV produced in human liver cells
(Figure 3C).9,25 In contrast, only very low infectivity was
found with the GFP control cells, corroborating the limiting amounts of endogenous (m)apoE expressed in
Hep56.1D cells.
For direct comparison of HCVTCP produced in mouse or
human hepatic cells, we developed the same trans-complementation system with Huh7.5 cells (cell line Huh7.5sgJFH-CNS2). Infectivity titers attained with this cell line
were well comparable with those of mouse cells demonstrating the high assembly competence of Hep56.1D
mouse liver cells (Supplementary Figure 6).
Biophysical properties of mouse and human cell-derived HCVTCP were determined by ultracentrifugation as
described above. The density profile of viral RNA of both
samples was comparable (Figure 3D). Core protein distribution across the gradient was also comparable, although
an additional peak was detected at very low density (Figure 3E), which was more pronounced when analyzing
infectivity (Figure 3F), arguing that either human cells are
more competent in producing lipoproteins or that huHCVTCP have a higher competence to associate with lipids.
In summary, these results demonstrate that infectious
muHCVTCP particles share similar although not identical
biophysical properties with HCV produced in human liver
cells.
ApoE Is a Component of Mouse Cell-Derived
Infectious HCV Particles
Because apoE is required for HCV infectivity and
assembly and an integral component of infectious HCV
particles,9,10 we wondered whether the same holds true for
mouse cell-produced particles. MuHCVTCP particles were
generated in cells transiently expressing HA-tagged (h)apoE3 or (m)apoE and subjected to HA-specific affinity
purification. Only tagged apoEs were detected in the immunocomplexes even though untagged proteins were expressed to comparable level (Figure 4A). Importantly, infectious particles were detected in immunocomplexes
obtained with supernatants of HA-apoE transfected cells
(Figure 4B). Thus, similar to human hepatocyte-derived
HCV, mouse cell-derived HCV particles are tightly associated with apoE residing on the surface of the particles.
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Figure 3. Biophysical properties of muHCVTCP particles. (A–C) Hep56-sgJFHcl3-CNS2 cells were transfected with constructs specified in the top.
Forty-eight hours later, culture supernatant was collected, concentrated by ultrafiltration, and fractionated by density gradient centrifugation. Ten
fractions were harvested from each gradient, and amounts of HCV RNA (A), core protein (B), and viral infectivity (C) contained in each fraction were
determined. (D–F) muHCVTCP contained in supernatant of Hep56-sgJFHcl3-CNS2 cells transiently expressing (m)apoE and huHCVTCP released from
Huh7.5-sgJFH-CNS2 cells expressing (h)apoE were analyzed by ultracentrifugation. Amounts of HCV RNA (D), core protein (E), and viral infectivity
(F) contained in each fraction were determined as above. Representative data of at least 2 independent experiments are shown.
Equal Entry Factor Dependency of Mouse and
Human Cell-Derived HCV Particles
Entry of HCV into human hepatocytes requires
multiple cell surface molecules, including CD81,28 scavenger receptor class B type I (SCARB1),29 Claudin 1,30 and
Occludin.16 To test whether HCV particles produced in
mouse cells utilize these entry molecules to the same
extent, neutralization experiments were conducted. Entry
of human and mouse cell-derived HCV particles was
blocked with comparable efficiency by all 3 tested antibodies, whereas matching control antisera had no effect
(Figure 5). We therefore concluded that entry receptor
dependency of HCV particles derived from human or
mouse cells is comparable. This result argued against
mouse cell-specific modifications of HCV particles affecting their entry receptor usage.
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MMH-sgJFH-CNS2. Amounts of HCV RNA in these cell
lines were comparable with those in Hep56.1D-derived
cells (Figure 6A). Endogenous amount of (m)apoE was
below the detection limit, but robust expression was obtained upon transfection with (h)apoE3 or (m)apoE expression constructs (Figure 6B). None of the transduced
genes affected HCV RNA replication as determined by
NS5A-specific Western blot, and core protein amounts
were comparable in all cell clones. Infectious HCV particles were not detectable in either of the cell lines transfected with the GFP control vector (Figure 6C). However,
infectivity was consistently detected in the supernatant of
MMH1-1- and AML12-derived cells expressing either the
human or mouse apoE gene. In agreement with the results obtained with Hep56.1D cells, infectivity titers were
not affected by the species origin of the apoE gene.
Whereas the titers attained with the MMH1-1 cells were
well comparable with those achieved with Hep56.1D cells,
infectivity titers achieved with AML12-derived cells were
about 10-fold lower, arguing for possibly limiting factors
of HCV assembly and release. Nevertheless, the fact that 3
different mouse liver-derived cell lines support production of infectious HCV particles provides compelling evidence that HCV assembly competence is a general property of mouse hepatocytes.
Discussion
To establish an immunocompetent fully permissive mouse model that is urgently required to study HCVspecific immune responses and pathogenesis, multiple
species restrictions need to be overcome. HCV entry into
mouse cell lines is possible upon ectopic expression of
HCV Assembly Competence Appears to be a
General Property of Mouse Liver Cells
So far, all our analyses have been based on the
mouse hepatoma cell line Hep56.1D. To determine
whether assembly competence is a unique property of this
cell line or of mouse liver cells in general, we established
the analogous trans-complementation system by using 2
other mouse cell lines containing a stably replicating
subgenomic HCV replicon: AML12-sgJFH1 and MMH11-sgJFH1.20 Both cell lines were transduced to express core
to NS2, resulting in cell lines AML12-sgJFH-CNS2 and
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Figure 4. Association of apoE with muHCVTCP particles. (A) ApoEspecific Western blot analysis of total proteins from Hep56-sgJFHcl3CNS2 cells transfected with (h)apoE3-, (h)apoE3-HA- (m)apoE-, or (m)apoE-HA-expression constructs. Input and immuno-captured proteins
are shown in the left and right panels, respectively. Molecular sizes are
indicated on the left of the blots, and proteins are specified on the right.
(B) Infectivity determination of immunocaptured muHCVTCP particles.
Equivalent amounts of infectious particles were subjected to HA affinity
capture as described in the Materials and Methods section. Captured
particles were eluted with an HA peptide and infectivity titers in input and
the corresponding eluates were determined by using limiting dilution
assay. Shown are the means of 3 independent experiments; error bars
represent standard deviations from the means.
Figure 5. Entry factor dependency of muHCVTCP particles. Huh7.5 cells
were inoculated with muHCVTCP (released from Hep56-sgJFHcl3-CNS2
cells overexpressing (m)apoE) or huHCVTCP particles (released from
Huh7.5-sgJFH-CNS2 cells) for 1 hour in the absence or presence of
antibodies specified in the bottom. Cells were washed 3 times with fresh
medium and incubated for 72 hours. Infectivity titers were assessed by
counting HCV-positive foci detected by immunohistochemical staining.
Mock incubation of virus samples served as negative control and was
used for data normalization (set to 100%). Shown are the means of 3
independent experiments; error bars represent standard deviations from
the means.
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Figure 6. Assembly of muHCVTCP particles in mouse liver cell lines AML12 and MMH1-1 cells. (A) Quantification of intracellular HCV replicon RNA
by using quantitative reverse-transcription polymerase chain reaction. HCV RNA copy number in 105 mouse cells specified in the bottom was
determined. (B) Expression analysis of HCV proteins and apoEs in MMH1-1 and AML12-derived cell lines by using Western blot. Cells were harvested
48 hours after transfection; mock transfected cells served as negative control. Molecular sizes are indicated on the left, and proteins are specified on
the right. (C) Infectivity released into supernatants of cells transfected with apoE expression constructs specified in the bottom was determined using
TCID50 assay. Shown are the means of 3 independent experiments; error bars represent standard deviations from the means.
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CD81 and occludin or by using a mouse CD81-adapted
HCV genome.16,17 Whereas HCV internal ribosome entry
site (IRES) activity in mouse and human cells appears to
be comparable,18,19 viral RNA replication is very inefficient
and requires selection approaches.20,21 Transient replication that mimics more closely an infection is virtually
undetectable, although the ectopic expression of microRNA-122 and knockout of interferon regulatory factor
3 or protein kinase R appears to increase RNA replication.31,32 Thus, HCV replication might be limited both by
intrinsic and induced restriction factors but also by low
expression and/or mouse/human incompatibility of important proviral factors. Owing to this insufficient RNA
replication, it could not be studied whether mouse cells
support production of infectious HCV particles. To overcome this limitation, we devised a trans-complementation
system with multiple mouse liver cell lines. We demonstrate that mouse cells do support assembly and release of
infectious HCV particles as long as sufficient levels of
apoE are expressed. In addition, we found that (1) each
human apoE isoform and mouse apoE support particle
production to the same extent and (2) (m)apoE supports
assembly also in human cells (Supplementary Figure 7).
Thus, apart from apoE, host factors contributing to HCV
particle production in mouse cells are neither limiting in
abundance, nor do they critically determine species restriction. However, as exemplified by the different expression levels of apoE in mouse hepatic cell lines and mouse
hepatocytes in vivo, we can not exclude the possibility that
other host factors affecting HCV assembly might limit
particle production in vivo.
Accumulating reports show that HCV morphogenesis is
tightly associated with lipoproteins and virions are re-
leased as unique lipoviro particles.5 A hallmark of this
assembly process is its dependence on apoE.9 –12 In this
study, we observed the same for mouse cell-derived HCV
particles corroborating the similarity of HCV assembly in
human and mouse cells. Interestingly, Hep56.1D cells
containing the subgenomic JFH-1 replicon expressed
much lower amounts of apoE as compared with naïve
cells. Moreover, also in the replicon cell clones derived
from AML12 and MMH1-1, apoE expression was barely
detectable. The underlying reasons are not known, but
low apoE expression in mouse replicon cells might be an
epiphenomenon perhaps linked to host cell conditions
that are more favorable for viral replication.
HCVTCP produced in mouse and human cells share similar
properties. First, we found that infectivity of human or
mouse cell-derived HCVTCP has a broad density range, comparable with HCV produced in Huh-7 cells.9 We note, however, that a fraction of HCVTCP particles produced in human
cells appear to be associated with lipids to a higher extent
and that these particles have a high specific infectivity. Second, infectivity of HCV particles produced in human or
mouse liver cells depends on the same set of entry molecules.
Third, assembly of human and mouse cell-derived HCV
particles depends on apoE amounts, and both particle types
are associated with it.
ApoE on lipoprotein particles directly binds to the LDL
receptor (LDLR), which is important for clearance of
remnant lipoproteins. LDLR is also involved in HCV entry
presumably via interaction with apoE on the surface of
the virion.33 LDLR binding affinity to apoE is remarkably
isoform specific. In vitro, apoE3 and E4 bind with similar
affinity to LDLR, whereas apoE2 has ⬃2% of this binding
capability.34,35 In line with these results, Hishiki et al
reported that HCV particles produced in apoE2-expressing cells were about 75% less infectious than apoE3- or
apoE4-containing HCV.36 We found however, that HCV
infectivity is not affected by a particular apoE allele indicating that the apoE-LDLR affinity is not decisive for
infectivity, at least in Huh7.5 cells. Moreover, very recently
Cun et al also reported that human apoE2, E3, and E4 are
equally efficient in supporting infectious HCV production
in Huh7.5 cells.37 The reason for these discrepancies is not
clear, but differences between the used cell lines eventually
expressing different levels of LDLR as well as other apoE
receptors such as Heparan sulfate proteoglycans, lowdensity lipoprotein receptor-related protein 1 (LRP1), or
apoE receptor R2 (LRP8) might account for that.
Apart from enabling assembly studies in mouse cells, the
extremely low expression levels of apoE in Hep56.1D cells,
which mimics a functional knock-down, provides an important tool to dissect the role of individual apolipoproteins for
HCV assembly independent from apoE. Moreover, HAtagged apoEs are fully functional thus providing an additional tool to isolate and characterize HCV particles as well
as apoE-binding viral and host cell proteins.
In summary, we demonstrate that mouse liver cell lines
are competent for assembly and release of HCV particles
provided that sufficient levels of apoE are expressed. This
result closes another gap in our understanding of species
restriction of HCV. In this respect, the block of viral RNA
replication is most likely the only remaining hurdle that
needs to be overcome to establish a fully permissive
mouse liver cell-based HCV system. Without doubt, such
a system will pave the way for the development of an
immunocompetent HCV permissive mouse model.
Supplementary Materials
Note: To access the supplementary material
accompanying this article, visit the online version of
Gastroenterology at www.gastrojournal.org and at doi:
10.1053/j.gastro.2011.06.010.
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Received January 28, 2011. Accepted June 3, 2011.
Reprint requests
Address requests for reprints to: Ralf Bartenschlager, PhD,
Department of Infectious Diseases, Molecular Virology, Heidelberg
University, Heidelberg, Germany. e-mail:
[email protected]; fax: (49) 6221 564570.
GASTROENTEROLOGY Vol. 141, No. 3
Acknowledgments
The authors thank Ulrike Herian for excellent technical assistance;
Susan Uprichard for providing the replicon cell lines MMH1-1 and
AML12; Thomas Baumert for provision of SCARB1- and Claudin 1specific antibodies; Heinrich Wieland for apoE2, 3, 4 constructs;
Stephan Urban for providing mouse liver homogenates; Charles M.
Rice for Huh7.5 cells and the NS5A-specific antibody 9E10; Takaji
Wakita for provision of the original JFH1 clone; and the Nikon
Imaging Center at the University of Heidelberg for continuous support
and providing access to all necessary equipment.
M.P.W.’s present address is Applied Molecular Virology, Institute
Pasteur Korea, 696 Sampyung-dong Bundang-gu, Seongnam-si,
Gyeonggi-do, South Korea.
Conflicts of interest
The authors disclose no conflicts.
Funding
Supported by a grant from the Deutsche Forschungsgemeinschaft
(SFB/TRR77, Teilprojekt 1; to R.B. and V.L.; and TRR83, Teilprojekt
13; to R.B.), by a grant from the European Union (ERC grant contract
no. 233130), and by the Marie Curie Training Network EI-HCV,
contract no. MRTN-CT-2006-035599 EI-HCV.
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