Journal of General Virology (2015), 96, 311–321
DOI 10.1099/vir.0.070433-0
Regulation of core expression during the hepatitis
C virus life cycle
Muhammad Sohail Afzal,1,2 Khaled Alsaleh,1 Rayan Farhat,1
Sandrine Belouzard,1 Adeline Danneels,1 Véronique Descamps,3
Gilles Duverlie,3 Czeslaw Wychowski,1 Najam us Sahar Sadaf Zaidi,2
Jean Dubuisson1 and Yves Rouillé1
Correspondence
Yves Rouillé
1
[email protected]
2
Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204,
Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
Atta ur Rahman School of Applied Biosciences (ASAB), National University of Science and
Technology (NUST), Islamabad, Pakistan
3
EA4294, Unité de Virologie Clinique et Fondamentale, CHU d’Amiens,
University of Picardie Jules Verne, Amiens, France
Received 31 July 2014
Accepted 23 October 2014
Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural
component of the virion, but also as a regulator of the formation of assembly sites. In this study, we
observed that core is expressed later than other HCV proteins in a single viral cycle assay,
resulting in a relative increase of core expression during a late step of the viral life cycle. This
delayed core expression results from an increase of core half-life, indicating that core is initially
degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed
kinetics of core expression were also observed using heterologous expression systems. Core
stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was
correlated with its expression levels and its oligomerization status. Therefore in the course of a
HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome
during an initial replication step allows core to be synthesized at higher levels as a stable protein,
during the assembly step of the viral life cycle.
INTRODUCTION
Hepatitis C virus (HCV) is a small, enveloped, singlestranded positive RNA virus, belonging to the Flaviviridae
family. With about 160 million persons infected globally,
HCV is a major public health problem (Lavanchy, 2011).
For many years, studies of the molecular and cellular
aspects of its infection have been hampered by the lack of a
cell culture system. However, with the recent development
of a HCV cell culture (HCVcc) system, regulatory mechanisms of the HCV infection can now be investigated in the
context of a complete life cycle.
HCV genome contains a single ORF. Its translation leads to
the production of a polyprotein precursor, which is cleaved
into ten polypeptides by cellular and viral endopeptidases.
The N-terminal third of the polyprotein contains the
structural proteins core, the capsid protein, and E1E2, a
heterodimeric envelope glycoprotein. They are followed by
the non-structural proteins p7, NS2, NS3, NS4A, NS4B,
NS5A and NS5B (Lindenbach et al., 2007). Non-structural
One supplementary figure is available with the online Supplementary
Material.
070433 G 2015 The Authors
proteins NS3-4A, NS4B, NS5A and NS5B are necessary and
sufficient for replication of the genomic RNA (Lohmann
et al., 1999). On the other hand, the assembly and budding
of new infectious particles is likely to involve all the
structural and non-structural proteins (Bartenschlager
et al., 2011; Miyanari et al., 2007). Mechanisms regulating
the transition from the translation/replication step to the
assembly step of the HCV life cycle have remained elusive.
Among the viral proteins, core appears to be a major
organizer of the assembly step. Core recruits active replication complexes to the assembly site by interacting with
NS5A (Masaki et al., 2008; Miyanari et al., 2007). NS2 and
p7 also regulate the assembly step, by interacting with each
other and with envelope glycoproteins and non-structural
protein NS3 (Jirasko et al., 2010; Popescu et al., 2011;
Stapleford & Lindenbach, 2011).
To study mechanisms potentially regulating the transition
from replication to assembly, we investigated if proteins
specifically involved in assembly are expressed with the
same kinetics as those that are involved in both replication
and assembly. It is generally assumed that all HCV proteins
are expressed with similar kinetics, since all HCV proteins
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311
M. S. Afzal and others
are initially synthesized from a single precursor. However,
the expression of a protein actually results from the balance
between its synthesis and degradation. By measuring the
kinetics of expression of HCV proteins in a single cycle
assay, we confirmed that most HCV proteins are expressed
with very similar kinetics. However, we also unexpectedly
observed a time lag for core expression.
RESULTS
Core is expressed later than other HCV proteins
during the HCV life cycle
To determine the kinetics of expression of HCV proteins,
we measured their relative expression levels at different
time points in a single cycle assay using Huh-7w7, CD81deficient Huh-7-derived cells (Rocha-Perugini et al., 2009).
We focused our study on proteins only involved in assembly (core, E1, E2 and NS2), and compared their expression
with that of two markers of replication complexes (NS3
and NS5A), which are involved in both replication
and assembly. As shown in Fig. 1(a), all proteins were
undetectable at 12 h post electroporation. Expression levels
rapidly rose between 12 and 36 h, to reach a maximal value
at 48 h. Then, at 72 h post electroporation, we observed an
apparent decrease of expression for all HCV proteins. This
apparent decrease can be attributed to the fact that infected
cells (24±1 % in these experiments) probably grew more
slowly than uninfected cells. Accordingly, the total amounts
of HCV proteins per well did not decrease at 72 h post
electroporation, when values were not normalized to the
protein content of the lysates (not shown). These results
indicate that HCV protein expression levels reached
equilibrium after approximately 36–48 h of expression.
Core expression levels appeared to increase less rapidly
than those of all other HCV proteins tested (Fig. 1a). Most
HCV proteins were found to accumulate with a halfmaximal value of 25–27 h (Fig. 1b). In contrast, the halfmaximal accumulation of core was reached at approximately 33 h, indicating a 6–8 h lag. To rule out that this
delay in core expression could in fact result from different
sensitivities of the antibodies used for quantification (Fig.
S1, available with the online Supplementary Material), we
repeated the quantification with a series of antibodies to
core, E1 and E2. Although some variations were observed
for a protein probed with different antibodies, the results
confirmed the delay of core expression (Fig. 1c). When
core and NS3 were quantified by ELISA, core reached
higher expression levels than NS3 at 48 h, and then
decreased at 72 h, whereas NS3 levels did not (Fig. 1d).
Compared with their expression at 48 h, the relative
expression at 24 h was lower for core (13 %) than for NS3
(29 %), consistent with results obtained by immunoblot
quantification. The percentage of secreted core protein
increased over time (Fig. 1e), and correlated with infectious
titres (Fig. 1f). However, core release was less than 1 %,
312
indicating that the lower levels of core expression at 24 h
could not be explained by an excess of secretion.
To confirm the delay of core expression in an unbiased
manner, we introduced an HA epitope tag in core or in
NS5A, in order to compare core and NS5A expression with
the same antibody. The insertion of an HA tag in core or in
NS5A did not alter replication efficiency, as assessed by
immunoblot analysis of E1 expression (Fig. 2a). Again,
slower kinetics of core expression were observed using an
anti-HA antibody (Fig. 2a). The delay in core expression
was very similar to that observed with the untagged virus
(Fig. 2b).
To further confirm the kinetics of core expression by an
independent method, we analysed core expression by
immunofluorescence. Huh-7 cells were infected with
HCVcc, and double-labelled with antibodies to core and
NS5A, at 32 or 72 h post-infection (p.i.). These two time
points were chosen to have well-detectable signals for the
early time point (32 h) and to have a major part of the
population of infected cells at a late step of the HCV life
cycle for the late time point (72 h). The experiment was
also performed in electroporated Huh-7w7 cells. All coreexpressing cells also expressed NS5A, while a number of
cells expressing NS5A did not express any detectable core,
whichever time the cells were fixed (Fig. 3a). As shown in
Fig. 3(b), there were more than two times more doublepositive Huh-7 cells at 72 h p.i. than at 32 h p.i. Similar
results were obtained with an HCVcc chimera expressing
genotype 1a structural proteins (Fig. 3b). The number of
double-positive cells reached about 95 % at 72 h in Huh7w7 cells, and only about 50–55 % in Huh-7 cells (Fig. 3b).
This difference probably resulted from the fact that
infections are synchronized in Huh-7w7 cells and not in
Huh-7 cells. Therefore, the Huh-7 population contains
infected cells at different stages of the viral life cycle, when
most of the Huh-7w7 cells are at a late step. These results
are consistent with core being expressed with slower kinetics
than NS5A, regardless of the difference in sensitivity of the
two antibodies, otherwise similar percentages of doublelabelled cells would have been measured at both time points.
A delay in core expression was also visible in the labelling
pattern of HCV foci. At 72 h p.i., isolated foci often
displayed a few cells brightly stained with both anti-core
and anti-NS5A antibodies, surrounded by cells with weaker
labelling (Fig. 3c). This difference in labelling is likely to
reflect the occurrence of at least two rounds of infections:
the small group of brighter cells at the centre representing
the primary infected cell later divided by one or two mitoses,
and the cells at the periphery with weaker labelling probably
resulting from a second round of infection. Considering this
scenario of focus formation, we consistently observed that
the difference of labelling intensities between primary and
secondary infected cells was less important for NS5A than
for core (Fig. 3d). We quantified the relative expression
levels of both proteins in secondary infected cells as a
percentage of the signal measured for the primary infected
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Journal of General Virology 96
HCV core expression
Time (h)
24 36
12
(b)
48
72
(c)
120
120
C
E1
E2
NS2
NS3
NS5A
C
100
E2
NS2
Expression (%)
E1
80
C (pAb)
C (ACAp27)
C (2H9)
E1 (A4)
E1 (1C4)
E2 (3.11)
E2 (AP33)
E2 (A11)
E2 (pAb)
100
Expression (%)
(a)
0
60
40
80
60
40
NS3
20
20
NS5A
tubulin
7000
6000
(e)
5000
4000
3000
2000
1000
0
24h
48h
24
36
Time post-electroporation (h)
0.8
0.6
0.4
0.2
0
72h
0
48
(f)
1.0
Core
NS3
Core release (%)
Expression [fmol (mg protein)–1]
(d)
Tubulin
12
Infectious titre (log10 TCID50)
0
24h 48h 72h
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
12
24
36
Time post-electroporation (h)
48
24h 48h 72h
Fig. 1. Kinetics of expression of HCV proteins. (a) JFH1 genomic RNA was introduced by electroporation into CD81-deficient
Huh-7w7 cells. The expression of indicated viral proteins was monitored by immunoblot at the indicated time post
electroporation. (b) The signals were quantified and plotted against time. (c) Quantification with two anti-E1 antibodies (green),
four anti-E2 antibodies (blue), and three anti-C antibodies (red). Expression levels were expressed as percentages of the 48 h
value. (d) Quantification by ELISA of core and NS3 in cell lysates. (e) Quantification by ELISA of core release. (f) Infectious
titres. Values are means±SD of two experiments, each performed in duplicate.
(a)
E1 (NS5A-HA)
E1 (HA-core)
100
NS5A-HA
HA (NS5A)
E1
NS5A-HA
virus
Tubulin
HA (core)
E1
tubulin
HA-core
virus
Expression (%)
0
(b) 120
Time (h)
12 24 36 48 72
80
HA-core
60
40
20
0
12
24
36
48
Time post-electroporation (h)
Fig. 2. Kinetics of core and NS5A expression of HA-tagged viruses. (a) Genomic RNAs of HA-tagged viruses were
electroporated into Huh7w7 cells and the expression of HA-core, NS5A-HA, E1 and tubulin was monitored. (b) Quantification
of the signals. HA signals are means ±SD of two experiments, each performed in duplicates. E1 signals were quantified once.
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M. S. Afzal and others
(b) 100
90
80
70
60
50
40
30
20
10
0
72 h
NS5A-positive cells
expressing core (%)
(a) 32 h
H77
JFH1
Huh-7w7
72 hpi
32 hpi
(d) 80
Relative expression (%)
(c)
NS5A
70
60
50
40
30
20
10
0
Core
NS5A core
Fig. 3. Relative expression of core and NS5A at 32 and 72 h post-infection. (a) JFH1-infected Huh-7 cells were fixed and
processed for detection of core (green) and NS5A (red) by immunofluorescence at 32 or 72 h p.i. (b) Huh-7 cells were infected
with JFH1 or H77/JFH1 chimera and Huh-7w7 cells were electroporated with JFH1 RNA. Infected (NS5A-positive) cells
expressing core were quantified at 32 and 72 h p.i. Values are means±SD from three independent experiments. (c) Doublelabel image of a focus at 72 h p.i. Each fluorescent signal was colour-coded with the LUT indicated on the right side. (d)
Relative expression levels of core and NS5A in peripheral cells of HCV foci. Values are means±SD from 135 cells. Bars, 20 mm.
cells of 10 well-isolated foci. We found a fourfold higher
relative expression level of NS5A, as compared with core, in
secondary infected cells (Fig. 3d). Again, this observation
points to a delay in core expression, as compared with
NS5A. Together with the results of the kinetics of expression,
these data indicate that core is expressed with a time lag
during the HCV life cycle.
Core is stabilized at a late stage of the HCV
infection
All HCV proteins are co-translated as a single polyprotein,
and thus synthesized in equimolar amounts. To assess
whether the peculiar kinetics of core expression could
result from a variation of its stability as the HCV life cycle
progresses, we measured its half-life at two time points
post-infection. We made use of a cycloheximide (CHX)
chase assay to measure half-lives of core, and of E1 and
NS5A as controls. When the analysis was performed at 32 h
p.i., a sharp decrease in core expression levels was observed
(Fig. 4a). In contrast, the expression levels of E1 (Fig. 4b)
and NS5A (Fig. 4c) decreased more slowly during the
chase. Immunoblot signals were quantified and plotted
versus CHX chase times, in order to estimate HCV protein
half-lives. Core half-life was found to be approximately
314
95 min, whereas E1 half-life was almost 9 h and NS5A halflife was longer than 4 h (Fig. 4d). Strikingly, when the
analysis was repeated at a later time point (72 h p.i.), core
appeared much more stable than at 32 h p.i. (Fig. 4a).
There was a tenfold increase in core half-life, whereas E1
and NS5A half-lives were increased by less than two times
(Fig. 4d). Taken together, these results indicate that core is
initially unstable, and is much more stable later on. This
result indicates that core undergoes a stabilization process
during the HCV life cycle.
Core stabilization depends on expression levels
and correlates with oligomerization
We next investigated if the stabilization of core is restricted
to the context of an infection, or if it could also be observed
in heterologous expression systems. Core was expressed in
Huh-7 cells by transfection, together with E1E2 and GFP,
and the kinetics of expression of the proteins were analysed
by immunoblotting (Fig. 5a). Again, a lag was observed for
core expression, as compared with E1, E2 and GFP (Fig.
5b), suggesting that the delayed core expression results
from intrinsic properties of this protein.
To assess whether this delayed expression resulted from
stabilization, core half-life was measured at 20 and at 40 h
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Journal of General Virology 96
HCV core expression
1
2
4
8
(a) CHX (h) 0
32 h p.i.
72 h p.i.
4
8
(c) CHX (h) 0
32 h p.i.
10
0
2 4 6 8
CHX chase (h)
100
72 h p.i.
32 h p.i.
10
1
0
2
4
8
NS5A
Relative expression
72 h p.i.
32 h p.i.
1
72 h p.i.
E1
Relative expression
Core
Relative expression
2
72 h p.i.
100
1
1
2 4 6 8
CHX chase (h)
(d)
100
72 h p.i.
32 h p.i.
10
1
Half-life (min)
(a) CHX (h) 0
32 h p.i.
0
1000
32 h p.i.
72 h p.i.
800
600
400
200
2 4 6 8
CHX chase (h)
0
Core E1 NS5A
Fig. 4. Change in core stability during the HCV life cycle. JFH1-infected Huh-7 cells were incubated for the indicated periods
with cycloheximide (CHX, 100 mg ml”1) to block protein translation, and the expression levels of core (a), E1 (b) and NS5A (c)
were monitored by immunoblot at 32 and 72 h p.i. Expression levels at 32 h p.i. (black symbols) and at 72 h p.i. (white symbols)
were plotted against CHX chase time. (d) Protein half-lives at 32 and 72 h p.i. from at least three independent experiments
(mean±SD).
after transfection. These two time points were chosen
because core signal was easily detectable at 20 h and was
close to the maximal expression level at 40 h. As shown in
Fig. 5(c), core half-life was approximately 90 min at the
earlier time point, and increased about three times at the
Time (h)
20
40
(b) 120
60
Core
GFP
E2
80
60
40
20
0
E1
Half-life (min)
(d) 2500
2000
20 h
40 h
Core
E1
E2
GFP
100
(e)
0
20
40
Time (h)
5
Core
GFP
10
HA (core)
1000
500
0
m.o.i.
60
20 h
40 h
Lactacystin (mM)
0
1500
(c) 400
350
300
250
200
150
100
50
0
Half-life (min)
0
Expression (%)
(a)
later time point (275±15 min). In contrast, GFP half-life
did not significantly vary over time. This result indicated
that the stabilization process could indeed be observed in a
heterologous expression system, although the extent of core
stabilization was not as high as during an HCV infection.
Tubulin
2
10 50
Ad:Core
2
10 50
Ad:GFP
Fig. 5. Core stabilization in heterologous expression systems. (a) Core, E1, E2 and GFP were co-expressed by transfection in
Huh-7 cells, and their expression was monitored by immunoblot at the indicated time post-transfection. (b) The signals were
quantified and plotted against time. For each protein, the expression levels were expressed as percentages of the 60 h value,
and are means±SD of three experiments each performed in duplicate. (c, d) Core and GFP were expressed in Huh-7 cells by
transfection (c), or by recombinant adenovirus transduction at different m.o.i. (d), and their half-lives were measured using the
CHX chase assay at 20 or 40 h post-transduction (values are mean±SD, from at least three independent experiments). (e)
Transfected Huh-7 cells were treated with lactacystin for 8 h at 20 h post-transfection and the expression of core and tubulin
was monitored by immunoblotting.
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M. S. Afzal and others
Core is degraded by the proteasome (Shirakura et al., 2007;
Suzuki et al., 2001). To test if low amounts observed at
20 h resulted from proteosomal degradation, we incubated
core-expressing cells with lactacystin, a potent proteasome
inhibitor, for 8 h, and quantified core by immunoblot (Fig.
5e). Similar levels of core were observed in control and
treated cells, suggesting that core is not degraded by the
proteosome at early time points of expression.
The time-dependent stabilization of core suggests that it
undergoes a conformational change over time that makes it
more stable. The fact that core stabilization also depends
on the level of expression of the protein suggests that a
concentration-dependent process, such as oligomerization,
could drive the mechanism of stabilization. To test this
hypothesis, we determined the kinetics of core oligomerization. Core monomers and oligomers from transfected
cells were separated by ultracentrifugation (Fig. 6a). A
major part of core protein (92±11 %) was detected in
supernatants at 20 h post-transfection. In contrast, at later
time points, core partitioned mainly into pellets (65±20
and 87±6 % at 40 and 60 h, respectively) (Fig. 6b). More
than 96 % of the non-oligomeric protein GFP remained in
supernatants up to 60 h post-transfection. This indicates
that core is initially expressed as non-pelletable forms, such
as monomers, dimers, or small oligomers, and is later
included into larger, pelletable oligomeric forms. The
kinetics of core oligomerization and stabilization were very
similar, suggesting a role of core oligomerization in the
mechanism of stabilization.
Effect of non-structural proteins and lipid droplets
on core stabilization
Core interacts with NS5A, and this interaction is thought
to be crucial for HCV particle assembly (Masaki et al.,
2008; Miyanari et al., 2007). To test the impact of NS5A,
and more generally of non-structural proteins, on core
stabilization, core was expressed in Huh-7 cells containing
a subgenomic replicon. To verify that NS5A actually
interacts with core in replicon-containing cells, we first
checked whether core was able to recruit replication
complexes to the surface of lipid droplets (LDs) in coretransfected, replicon-containing cells, as it does in JFH1infected cells (Miyanari et al., 2007). As reported previously
for a genotype 1b replicon (Shi et al., 2003), NS5A was not
associated to LDs in cells harbouring a JFH1 subgenomic
replicon in the absence of core expression (Fig. 7a). In
contrast, when core was expressed, NS5A labelling was
almost completely redistributed to the vicinity of LDs,
where core was also localized (Fig. 7a). This core-mediated
recruitment strongly suggests that interactions between
core and NS5A occur in replicon-containing cells expressing core. Therefore, this experimental setting was relevant
for investigating the impact of non-structural proteins on
core stability. Similar values of core half-life were obtained
in naı̈ve and replicon-containing cells, both at 20 h
(90±12 min in Huh-7 cells; 109±18 min in replicon cells),
and at 40 h post-transfection (269±18 min in Huh-7 cells;
304±27 min in replicon cells) (Fig. 7b). These data indicate
that the presence of non-structural proteins and the active
replication of viral RNA have no impact on core half-life, or
on the mechanisms that lead to core stabilization.
Core also interacts with LDs, and this interaction is
important for HCV assembly (Boulant et al., 2007). To test
if LDs have any influence on core stability, we treated
(a)
20 h
20 h
40 h
60 h
(Overexposed)
P S P S P S P S P S P S P S P S
Core
GFP
(b) 100
90
80
70
60
50
40
30
20
10
0
Oligomerization (%)
Then, to test the impact of core expression levels on core
half-life, we made use of an adenoviral vector. Huh-7 cells
were transduced with recombinant defective adenoviruses
expressing HCV core, or GFP as a control, at different
multiplicities of infection, in order to express core and GFP
at different levels, and their half-lives were measured 20 h
or 40 h later. The half-life of core did not significantly vary
in 20 h samples (from 85±7 to 101±10 min). In contrast,
we observed a dramatic increase of core half-life (from
171±16 to 2108±103 min) in 40 h samples (Fig. 5d),
corresponding to 2- to 21-fold increases of core half-life,
depending on adenovirus m.o.i. GFP half-life was only
slightly longer in 40 h samples compared with 20 h
samples (1.2-fold increase), but did not vary with the
expression levels of the protein. These results indicate that
core is stabilized in a manner dependent on its time and
level of expression.
Core
GFP
20 h
40 h
60 h
Fig. 6. Kinetics of core oligomerization. (a) Core and GFP were co-expressed by transfection in Huh-7 cells. At the indicated
times post-transfection, cells were lysed and the post-nuclear supernatants were centrifuged at 100 000 g for 30 min. The
expression of core and GFP in pellets (P) and supernatants (S) was monitored by immunoblot. (b) Quantification of protein
contents in pellets (values are mean±SD of three experiments each performed in duplicate).
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Journal of General Virology 96
HCV core expression
(a)
NS5A
Core
Merge
(d) 600
(c)
20 h
40 h
Half-life (min)
500
Half-life (min)
(b) 400
350
300
250
200
150
100
50
0
LD
Mock
Oleic acid
20 h
40 h
400
300
200
100
0
Mock Oleic
acid
Huh-7 SGR Huh-7 SGR
Core
GFP
Fig. 7. Effect of non-structural proteins and oleic acid treatment on core stability. (a) Core was expressed by transfection in
subgenomic replicon-containing Huh-7 cells, and the cells were processed for immunofluorescence detection of core (red),
NS5A (blue) and LDs (green). Each channel is shown in grey levels for better visualization and in colour in the merged image.
The star indicates a cell expressing core. Bar, 20 mm. (b) Core and GFP were expressed by transfection in Huh-7 cells or in
subgenomic replicon-containing cells (SGR), and their half-lives were measured using the CHX chase assay at 20 or 40 h
post-transfection (values are mean±SD, from at least three independent experiments). (c) Huh-7 cells were incubated for 24 h
in culture medium containing 100 mM oleic acid. LDs and nuclei were stained with BODIPY 493/503 and DAPI, respectively.
Bar, 20 mm. (d) Core was expressed by transfection in Huh-7 cells and the cells were treated or not with 100 mM oleic acid.
Core half-life was measured using the CHX chase assay at 20 or 40 h post-transfection (values are mean±SD, from at least
three independent experiments).
core-expressing cells with oleic acid (OA). This treatment
resulted in an increase of both the number and the size of
LDs (Fig. 7c). Image analysis revealed that mean LD size
increased 2.6-fold and their number per cell about 1.5-fold
(data not shown), resulting in an increase of total LD
surface per cell of approximately four times. In the
presence of OA, core half-life was about two times longer,
both at 20 h (92±2 min without OA; 174±19 min with
OA) and at 40 h (252±18 min without OA; 484±29 min
with OA), suggesting that LDs have a positive influence on
core stability (Fig. 7d). It is worth noting that the treatment
with OA, although positively affecting core half-life at both
time points, had no effect on the extent of core stabilization
(about 2.8-fold, in these experiments). These data suggest
that LDs have a positive effect on core half-life, but no
major contribution to the mechanism leading to core
stabilization.
DISCUSSION
For many viruses, progress through the life cycle is
controlled by the successive expression of different sets of
viral proteins over time. Many DNA viruses selectively
express proteins during different steps of their life cycle,
through transcriptional regulation of immediate early,
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early and late genes. For some RNA viruses, the regulation
of protein expression occurs through subgenomic RNA
production or translational frameshifting. For Flaviviridae
family members, these two types of regulatory processes
have not been observed, except for frameshifting events in
the core coding sequence of HCV (Walewski et al., 2001;
Xu et al., 2001). However, the functional importance of
this frameshifting event in the regulation of the life cycle of
the virus has not been clearly established (McMullan et al.,
2007; Vassilaki et al., 2008).
We now describe a new mode of selective protein
expression for HCV core, through the stabilization of the
protein. Since protein expression is the result of a balance
between synthesis and degradation, and since all HCV
proteins are initially synthesized in equimolar amounts,
differences in expression kinetics likely result from
differential stability. In our study, expression levels were
normalized to their value at 48 h post-electroporation.
Therefore, proteins with similar expression kinetics did not
necessarily reach equivalent absolute expression levels.
Nevertheless, all these proteins have equivalent relative
expression kinetics, indicating that their degradation rates
are constant, or at least do not significantly vary over time.
Accordingly, we observed similar E1 and NS5A half-lives at
both time points. This indicates that the expression profiles
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317
M. S. Afzal and others
of most HCV proteins are likely to reflect a steady accumulation in relation to the amplification of the genomic
RNA in infected cells.
In contrast, the peculiar kinetics observed for core indicate
that its expression involves different mechanisms. Such a
difference in kinetics could in fact result from several
potential mechanisms. Post-translational modifications
of core at early time points could result in a lack of
recognition by antibodies. However, this possibility is very
unlikely, since the same delay was observed with viruses
expressing tagged proteins. A differential usage during the
HCV life cycle of an internal translation initiation site
which was recently identified (Eng et al., 2009) could also
result in an apparent delayed core expression. However, the
antibodies we used do not allow testing of this possibility.
Our results on core half-life led us to favour the hypothesis
that the stability of core varies over the course of the HCV
life cycle, as the mechanism likely to be responsible for its
delayed expression.
Core has been previously shown to be an unstable protein in
heterologous expression systems (Shimoike et al., 2006;
Shirakura et al., 2007; Suzuki et al., 2001). Accordingly, we
measured a half-life of about 90–100 min in this study,
which indeed corresponds to a rather high turnover for a
membrane protein. Interestingly, similar half-life values were
obtained for core in heterologous expression systems and in
HCV infected cells at early time points, indicating that
the unstable nature of core protein is not restricted to
heterologous expression systems. At later time points, core
was found to be much more stable, and this stabilization was
dependent on its levels of expression. We did not observe any
impact of HCV non-structural proteins, again suggesting
that this phenomenon mainly relies on intrinsic properties of
the core protein, rather than on cues from other viral factors.
Core has been shown to be ubiquitylated by ubiquitin
ligase E6AP and degraded by the proteasome (Shirakura
et al., 2007; Suzuki et al., 2001). However, we did not
observe any stabilization of core or any accumulation of
ubiquitylated core in cells treated with lactacystin or
MG132, two proteasome inhibitors, at early time points of
core expression (Fig. 5e and data not shown). Indeed, this
lack of action of proteasome inhibitors on core expression
has already been reported by others (Hope & McLauchlan,
2000; McLauchlan et al., 2002). This suggests that this
degradative pathway is not the one that operates at the
early time points of core expression.
The time and concentration dependence of core stabilization suggests that core interferes with its own degradation.
It is unlikely that core inhibits proteosomal activity,
because long-term inhibition of the proteasome is toxic
for the cells. Therefore it is more likely that the interference
with its own degradation results from a post-translational
modification that makes core less sensitive to degradation.
As this post-translational modification appears to be
concentration-dependent, an oligomerization-dependent
process is the most likely. In support of this hypothesis,
318
we observed a good correlation between core oligomerization and stabilization. Previous reports indicated that core is
a protein prone to oligomerize in infected and transfected
cells (Alsaleh et al., 2010; Klein et al., 2004; Kunkel et al.,
2001; Majeau et al., 2004). Core is known to form dimers
(Boulant et al., 2005), and is usually found in cell lysates and
cell-free expression systems as a mixture of monomers/
dimers and larger oligomers, which can be resolved by
ultracentrifugation (Alsaleh et al., 2010; Klein et al., 2004).
Although the dissociation constants relative to core oligomerization are unknown, it can be reasonably speculated
that they are in a range that allows core to be in equilibrium
between mono/dimeric and larger oligomeric forms inside
the cell. Consequently, higher expression levels should drive
more core protein towards oligomeric structures. Therefore,
the stabilization of core can be interpreted as a transition
from a mono/dimeric less stable form to an oligomeric,
more stable form of the protein. In a cell-free expression
system, core oligomers were previously shown to be more
resistant to proteolysis than core monomers (Klein et al.,
2004). By analogy, core oligomers would be the stable form
of the protein in infected cells, whereas core monomers
would be preferentially recognized and degraded by cellular
quality control systems. Alternatively, we cannot exclude the
possibility that core stabilization could actually result not
directly from oligomer formation, but from another
putative post-translational modification that would preferentially occur on core protein engaged in oligomers. We
also cannot exclude that core, which is a membraneassociated protein, could partition over time in detergentresistant microdomains of the ER membrane, and that its
presence in pellets after ultracentrifugation reflects such an
accumulation in insoluble membranes.
Core is obviously required during late stages of the HCV life
cycle, when virion assembly occurs. In addition to its role as a
structural component of the virion, core also orchestrates
the formation of assembly sites at the ER/LD interface
(Bartenschlager et al., 2011; Miyanari et al., 2007). An
optimal expression of core is thus essential during late steps
of the HCV life cycle, when assembly occurs. On the other
hand, subgenomic replicon studies have indicated that core is
not required for RNA replication (Lohmann et al., 1999), and
it has been shown that core deletion and point mutations
inhibiting HCV assembly have no impact on translation or
replication of the viral genome (Alsaleh et al., 2010; Murray
et al., 2007). All these data are in line with our finding that
core is actually expressed with a lag of 6–8 h, and is thus
expressed at low levels when replication begins. We propose
that this oligomerization-mediated core stabilization is an
adaptive mechanism that allows core to be expressed at
higher levels during a late step of the HCV life cycle, when it
is required for orchestrating HCV assembly.
METHODS
Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), PBS, FCS,
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene
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Journal of General Virology 96
HCV core expression
(BODIPY 493/503) and DAPI were purchased from Life Technologies.
Protease inhibitor mix (complete) was from Roche. Other chemicals
were from Sigma.
(Mirus). Core and GFP-expressing plasmids were co-transfected in a
9 : 1 ratio.
Adenoviruses. The coding sequence of JFH1-CSN6A4 core residues
Antibodies. Mouse anti-E1 mAb A4, anti E2 mAb A11 (Dubuisson
et al., 1994) and rat anti-E2 mAb 3/11 (Flint et al., 1999) were produced
in vitro by using a MiniPerm apparatus (Heraeus). Mouse anti-core
ACAP-27 (Maillard et al., 2001) and anti-NS3 (486D39) mAbs were
provided by J.-F. Delagneau (Bio-Rad). ACAP-27 epitope was mapped
to residues 40–53 (Maillard et al., 2001). Rabbit polyclonal anti-E2 and
mouse anti-core mAb 2H9 (IgG1) (Wakita et al., 2005) were provided
by T. Wakita (National Institute of Infectious Diseases, Tokyo, Japan).
2H9 mAb epitope was mapped to residues 29–36 (T. Wakita, personal
communication). Mouse anti-E2 mAb AP33 (Clayton et al., 2002) and
rabbit anti-core antiserum R526, raised against core residues 1–59
(Clayton et al., 2002), were provided by A. H. Patel (Institute of
Virology, Glasgow, UK). Sheep anti-NS5A antiserum (Macdonald et al.,
2003) was provided by M. Harris (University of Leeds, UK). Mouse
anti-NS5A mAb 9E10 (IgG2a) (Lindenbach et al., 2005) and anti-NS2
mAb 6H6 (Dentzer et al., 2009) were provided by C. M. Rice (the
Rockefeller University, NY, USA). Human anti-E1 mAb 1C4 was
obtained from Innogenics. Mouse anti-b-tubulin mAb was from Sigma.
Mouse anti-GFP and rat anti-HA (3F10) mAbs were from Roche.
Cell culture. Huh-7 (Nakabayashi et al., 1982) and Huh-7w7
(Rocha-Perugini et al., 2009) cells were grown in DMEM supplemented with glutamax-I and 10 % FCS.
HCVcc. The virus JFH1-CSN6A4 used in this study contained cell
culture adaptive mutations and a reconstituted A4 epitope in E1
(Goueslain et al., 2010).
A virus with a tagged core was constructed by inserting a tag
(YPYDVPDYAI) containing an HA epitope (underlined sequence)
between residues Ser 2 and Thr 3 of core. To insert an HA tag in NS5A,
unique BglII and NruI sites were first inserted by overlapping PCR
between codons of Pro 418 and Leu 419. Synthetic oligonucleotides
59-GATCTTATCCATACGATGTTCCAGATTACGCGATATCG-39 and
59-GCTATAGCGCATTAGACCTTGTAGCATACCTATT-39 were annealed
and ligated into BglII and NruI sites. The sequence inserted between
NS5A residues 418 and 419 was RSYPYDVPDYAISR. The constructs
were verified by sequencing. The H77/JFH1 chimera, which incorporates the coding sequence of core to NS2 of H77 origin into JFH1, was as
previously described (Maurin et al., 2011).
Viral genomic RNAs were produced and delivered into Huh-7 or
Huh-7w7 cells by electroporation as described by Kato et al. (2003).
For infection assays, subconfluent Huh-7 cells grown in a P24-well
were incubated with HCVcc in 200 ml of medium for 2 h, and the
inoculum was replaced with fresh culture medium. For the single
cycle assay, 46106 Huh-7w7 cells were electroporated with 10 mg of
in vitro transcribed viral RNA, and plated into 6-well clusters.
Subgenomic replicon. The plasmid pSGR-JFH1 (Kato et al., 2003)
was processed as described for HCVcc. Electroporated cells were
selected with 0.5 mg geneticin ml21.
1–191 was introduced in a recombinant defective adenovirus vector
(Ad:core) by homologous recombination in Escherichia coli, as
described by Séron et al. (2011). The recombinant adenovirus
expressing EGFP was described previously (Séron et al., 2011). Viral
stocks were titred in Huh-7 cells by the TCID50 method using
immunofluorescence or GFP fluorescence to detect positive cells.
Immunoblotting. Cells were rinsed three times with cold PBS, and
lysed at 4 uC for 20–30 min in a lysis buffer containing 50 mM Tris/
HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 % (v/v) Triton-X, 0.1 %
(w/v) SDS, 1 mM PMSF, and a mix of protease inhibitors. The
protein content was determined by the bicinchoninic acid method
(Sigma). The signals were recorded using a LAS 3000 apparatus
(Fujifilm). Quantification of unsaturated signals was carried out using
the gel quantification function of ImageJ.
Cycloheximide (CHX) chase assay. Cells were incubated with
100 mg CHX ml21 for up to 8 h. Proteins were quantified by
immunoblotting. Proteins expression levels were plotted against time,
and the half-life was calculated from the decay constant of the fitted
exponential curve.
Core oligomerization. Cells were lysed at 4 uC for about 20 min in
lysis buffer without SDS. Cell lysates were centrifuged at 600 g, and
post-nuclear supernatants were centrifuged at 100 000 g and 4 uC
for 30 min. High-speed supernatants and pellets were analysed by
immunoblotting.
Immunofluorescence. Indirect immunofluorescence labelling was
performed as previously described (Rouillé et al., 2006). LDs were
stained with BODIPY 493/503 (0.5 mg ml21; Invitrogen).
ELISA. Core was quantified by a fully automated chemiluminescent
microparticle immunoassay (Architect HCVAg, Abbott; Descamps
et al., 2012). NS3 was quantified with a kit from BioFront Technologies.
ACKNOWLEDGEMENTS
The authors thank Dr Ekaterina Kolesanova for helpful discussions.
M. S. A. thanks Dr I. Qadri for his support. We are grateful to C. M.
Rice, T. Wakita, J.-F. Delagneau, A. Patel and M. Harris, for providing
us with very valuable antibodies, and D. Monté for the GFPexpressing adenovirus. Some data were generated with the help of the
imaging core facility of the Institut Pasteur de Lille (BICeL). This
work was supported in part by the French Agence Nationale de
Recherche sur le Sida et les hépatites virales (ANRS). M. S. A. was
supported by a pre-doctoral fellowship from the joint split PhD
scholarship programme of the Higher Education Commission and the
French embassy in Pakistan. S. B. is supported by a Marie Curie
International Reintegration Grant (PIRG-GA-2009-256300).
Plasmids. To construct a core expression plasmid, a cDNA encoding
residues 1–191 with a HA tag (AYPYDVPDYADI) inserted between
residues 1 and 2 was subcloned into pCIneo (Promega) between a
Kozak consensus sequence and a stop codon. The construct was
confirmed by sequencing. The plasmid used to express E1E2
glycoproteins (pCDNA-E1E2) contained the E1E2 coding sequence
of the JFH1 strain. The GFP-expression plasmid (pEGFP-C1) was
from Clonetech.
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