Journal of General Virology (2013), 94, 2221–2235
DOI 10.1099/vir.0.053868-0
Analysis of hepatitis C virus core/NS5A protein
co-localization using novel cell culture systems
expressing core–NS2 and NS5A of genotypes 1–7
Andrea Galli, Troels K. H. Scheel, Jannick C. Prentoe, Lotte S. Mikkelsen,
Judith M. Gottwein and Jens Bukh
Correspondence
Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical
Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International
Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen, Denmark
Jens Bukh
[email protected]
Received 26 March 2013
Accepted 24 July 2013
Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent
of infectious cell culture systems, the HCV particle assembly and release processes are finally
being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets
(cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current
knowledge on assembly and release is primarily based on studies in genotype 2a cell culture
systems; however, given the high genetic heterogeneity of HCV, variations might exist among
genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core–
NS2 and NS5A from genotypes 1–7, and analysed core and NS5A co-localization in infected
cells. Huh7.5 cells were transfected with RNA of core–NS2/NS5A recombinants and putative
adaptive mutations were analysed by reverse genetics. Adapted core–NS2/NS5A recombinants
produced infectivity titres of 102.5–104.5 f.f.u. ml”1. Co-localization analysis demonstrated that the
core and NS5A proteins from all genotypes co-localized extensively, and there was no significant
difference in protein co-localization among genotypes. In addition, we found that the core and
NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER
associated at later stages. Finally, we found that different genotypes showed varying levels of
core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we
developed a panel of HCV genotype 1–7 core–NS2/NS5A recombinants producing infectious
virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different
genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions
during assembly and release of HCV particles of all major genotypes.
INTRODUCTION
Hepatitis C virus (HCV) is a major cause of severe liver
disease such as chronic hepatitis, liver cirrhosis and
hepatocellular carcinoma (Alter & Seeff, 2000). Due to its
high degree of genetic diversity, HCV has been classified
into seven major genotypes and several subtypes based on
sequence homology (Bukh et al., 1993; Simmonds et al.,
1993, 2005). The virus particle contains a positive-sense
ssRNA genome, which encodes a single ORF flanked by 59and 39-untranslated regions (UTRs). The ORF encodes a
polyprotein that is processed by viral and cellular proteases
to produce structural proteins (core, E1 and E2), p7 and
non-structural proteins (NS2, NS3, NS4A/B and NS5A/B)
(Gottwein & Bukh, 2008). The entire viral life cycle of HCV
could not be studied in vitro until 2005, when the first cell
Two supplementary figures and seven tables are available with the
online version of this paper.
053868 G 2013 SGM
culture system based on the genotype 2a strain JFH1 was
developed (Wakita et al., 2005). J6/JFH1 recombinants,
produced by replacing the core–NS2 region of JFH1 with the
corresponding region derived from the genotype 2a J6CF
isolate (Yanagi et al., 1999), produced higher infectivity
titres and did not rely on culture adaptation (Gottwein et al.,
2007; Lindenbach et al., 2005; Pietschmann et al., 2006).
Several other JFH1-based recombinants, carrying the core–
NS2 region from the seven major genotypes, were subsequently developed (Gottwein et al., 2007, 2009; Jensen et al.,
2008; Pedersen et al., 2013; Pietschmann et al., 2006; Scheel
et al., 2008, 2011a; Yi et al., 2007). These constructs have
been useful for studying the effect of neutralizing antibodies
and antiviral drugs against diverse HCV genotypes, and for
functional studies of core–NS2 interactions (Gottwein et al.,
2009, 2011; Griffin et al., 2008; Pedersen et al., 2013; Prentoe
et al., 2011). In addition, we developed a panel of
recombinants carrying the NS5A region from genotypes
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A. Galli and others
1–7 in the J6/JFH1 backbone, with the purpose of analysing
the effect of newly developed NS5A inhibitors on different
strains of HCV and for functional genotype-specific studies
of NS5A (Scheel et al., 2011b, 2012). However, at the onset
of this study, efficient culture systems using recombinants
combining strain-specific structural proteins and NS5A were
only available for genotype 2a isolates (Scheel et al., 2011b;
Wakita et al., 2005; Zhong et al., 2005).
The availability of genotype 2a HCV cell culture systems
has allowed the study of essential steps of the viral
replication cycle, including viral assembly and release
(Bartenschlager et al., 2011; Lindenbach, 2013). To
produce infectious viruses, newly synthesized HCV genomes need to be delivered to the site of assembly, probably
the surface of cytoplasmic lipid droplets (cLDs) or the
cytosolic side of the endoplasmic reticulum (ER), and
packaged into the nucleocapsid (Appel et al., 2008;
Miyanari et al., 2007; Shavinskaya et al., 2007). The core
and NS5A proteins play essential roles in the early stage of
virion assembly (Masaki et al., 2008) and are among the
better-understood proteins involved in particle formation.
The core protein is the main constituent of the viral
nucleocapsid, which packages the genomic RNA for
particle release. NS5A is a phosphoprotein involved in
the formation of the viral replication complex and has been
shown to possess RNA-binding capacity (Huang et al.,
2005; Tellinghuisen et al., 2005), suggesting that it could be
involved in the transport of viral genomic RNA to the
assembly site (Miyanari et al., 2007). After synthesis, core
protein is mobilized to the surface of cLDs, where it is
thought to initiate particle assembly (McLauchlan et al.,
2002; Miyanari et al., 2007). The extent of core protein
accumulation on the surface of cLDs is inversely correlated
with the efficiency of particle assembly (Boulant et al.,
2007; Shavinskaya et al., 2007), probably reflecting a
transient localization of core protein on cLDs prior to its
transfer to the ER where the assembly process can proceed
(Boson et al., 2011). The core and NS5A proteins colocalize extensively in the cytoplasm and their interaction is
critical for the production of viral particles (Masaki et al.,
2008; Miyanari et al., 2007). Moreover, both localize on the
surface of cLDs and the extent of core/NS5A colocalization on cLDs is also inversely correlated with the
efficiency of virion production (Appel et al., 2008). NS2 is a
major player in the subsequent stages of assembly (Jirasko
et al., 2008; Ma et al., 2011; Popescu et al., 2011). The
nascent viral particle subsequently enters the ER lumen,
where it associates with E1/E2 through a not fully defined
mechanism and interacts with very-low-density lipoproteins to form lipoviroparticles (Gastaminza et al., 2008;
Huang et al., 2007). Mature virions are then released
through the secretory pathway via the Golgi network.
Several aspects of these processes remain unclear, and our
knowledge of the factors implicated and their interaction is
incomplete. Moreover, these processes have been only
elucidated in experiments with genotype 2a viruses, and the
genetic divergence between HCV isolates could result in
2222
variation in the assembly and release pathways among
different genotypes.
Here, we developed novel cell culture-adapted recombinants with genotype 1–7-specific core–NS2/NS5A in the
JFH1 backbone. We demonstrated that core and NS5A
proteins from all recombinants could be visualized by
immunofluorescence microscopy, and displayed similar
co-localization patterns irrespective of the HCV genotype.
The developed culture systems are suitable for investigation
of the assembly of HCV particles in the context of different
genotypes, since they carry isolate-specific versions of most
viral proteins involved in particle production and release.
RESULTS
Development of HCV recombinants carrying
genotype-specific core–NS2 and NS5A regions
Most functional studies of HCV in the context of the
complete viral life cycle have relied on genotype 2a cell
culture systems. To enable genotype-specific studies of
interactions between NS5A and the structural HCV
proteins for all seven major HCV genotypes, we developed
core–NS2/NS5A recombinants containing the core–NS2
and NS5A regions from prototype isolates of genotypes 1a,
1b, 3a, 4a, 5a, 6a and 7a (Bukh et al., 2010; Gottwein et al.,
2009; Sakai et al., 2007). Thus, we replaced the complete
NS5A of the following JFH1-based core–NS2 recombinants
that also carried culture-adaptive mutations with the
corresponding strain-specific NS5A consensus sequence:
H77C/JFH1V787A,Q1247L (1a), TN/JFH1R1408W (1a), J4/
JFH1F886L,Q1496L (1b), S52/JFH1I787S,K1398Q (3a), ED43/
JFH1T827A,T977S (4a), SA13/JFH1A1021G,K1118R (5a), HK6a/
JFH1F349S,N417T (6a) and QC69/JFH1L878P (7a) (all numbering is according to the H77 reference polyprotein,
GenBank accession no. AF009606) (Gottwein et al., 2007,
2009; Jensen et al., 2008; Scheel et al., 2008, 2011a).
Such core–NS2/NS5A recombinants could potentially produce higher titres compared with previously developed
genotype-specific core–NS2 and/or NS5A recombinants
(Gottwein et al., 2009; Scheel et al., 2011b), since efficient
virus production could depend on genotype-specific
interactions between NS5A and core.
In vitro-transcribed RNA corresponding to the HCV
genome of core–NS2/NS5A genotype 1–7 recombinants
was transfected into Huh7.5 hepatoma cells in parallel
with RNA of the genotype 2a recombinant, J6/JFH1
(Lindenbach et al., 2005). One day after transfection, for
most recombinants 10–30 % of cells expressed HCV core
protein, as determined by immunostaining. However, for
the ED43(4a), SA13(5a) and QC69(7a) recombinants, only
1–5 % of cells were positive (Fig. 1). J6/JFH1 infected most
of the culture after 3–6 days, while the same was observed
for S52(3a), HK6a(6a) and QC69(7a) after 6–12 days, and
for H77C(1a), TN(1a), J4(1b), ED43(4a) and SA13(5a)
after 18–39 days (Fig. 1). Only the QC69 recombinant
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Journal of General Virology 94
Co-localization of HCV core and NS5A of genotypes 1–7
75
50
25
0
0
4
8
12 16 20 24 28 32 36 40 44
Time p.t. (days)
(c)
75
50
25
0
0
4
8
12 16 20 24 28 32 36 40 44
Time p.t. (days)
J6/JFH1
S52(3a)I787S,K1398Q
S52(3a)I787S,K1398Q,C2419R
(d)
100
75
50
25
0
0
4
8
12 16 20 24 28 32 36 40 44
Time p.t. (days)
ED43(4a) (no mutations)
J6/JFH1
ED43(4a)T827A,T977S,Y1644H,E2267G
ED43(4a)T827A,T997S
(e)
Core-positive Huh7.5 cells (%)
Core-positive Huh7.5 cells (%)
100
H77(1a)V787A,C1185S,Q1247L
TN(1a)I1312V,R1408W
J4(1b)F886L,C1185S,Q1496L
100
75
50
25
0
0
4
8
12 16 20 24 28 32 36 40 44
Time p.t. (days)
J6/JFH1
SA13(5a) (no mutations)
SA13(5a)A1021G,K1118R SA13(5a)A1021G,K1118R,R1978G,C2419R
4
8
(f)
100
75
50
25
0
0
4
8
12 16 20 24 28 32 36 40 44
Time p.t. (days)
J6/JFH1
HK6a(6a)F349S,N417T
HK6a(6a)F349S,N417T,I2268N
Core-positive Huh7.5 cells (%)
Core-positive Huh7.5 cells (%)
100
J6/JFH1
H77(1a)V787A,Q1247L
TN(1a)R1408W
J4(1b)F886L,Q1496L
Core-positive Huh7.5 cells (%)
Core-positive Huh7.5 cells (%)
(b)
(a)
100
75
50
25
0
0
12 16 20 24 28 32 36 40 44
J6/JFH1
QC69(7a)L878P
Time p.t. (days)
Fig. 1. Evaluation of HCV antigen-positive Huh7.5 cells after transfection of JFH1-based recombinants with genotype-specific
core–NS2/NS5A. The percentage of positive cells in culture was evaluated by staining with anti-core antibody after transfection
with RNA transcripts of recombinants of isolates(genotypes): (a) H77C(1a), TN(1a) and J4(1b), (b) S52(3a), (c) ED43(4a), (d)
SA13(5a), (e) HK6a(6a) and (f) QC69(7a). Each panel contains data from one or more experiments and includes a
representative J6/JFH1 control. Selected recombinants were tested in multiple experiments. Numbering of mutations is
according to the H77 reference polyprotein (GenBank accession no. AF009606). p.t., Post-transfection.
produced infectivity titres above 103 f.f.u. ml21 during the
first 10 days after transfection, although delayed around
5 days compared with J6/JFH1 (Fig. 2). Since mutations
adapting the previously developed core–NS2 recombinants
could potentially compensate for interactions with JFH1
http://vir.sgmjournals.org
NS5A and could thus be a disadvantage for core–NS2/NS5A
recombinants with strain-specific NS5A, we tested ED43(4a)
and SA13(5a) core–NS2/NS5A without such mutations.
However, this led to no spread of infection for ED43(4a) and
slower spread of infection for SA13(5a) (Fig. 1).
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A. Galli and others
(b)
5
Infectivity titre [log10(f.f.u. ml–1)]
4
3
3
<2.3
(d)
5
8Q
K1
39
87
I7
,C
K1
S,
C
L,
96
14
Q
I7
F8
87
86
S,
L,
24
19
R
85
S
39
8Q
S52(3a)
11
14
Q
L,
86
L,
47
87
V7
(c)
J4(1b)
96
L
85
S
11
C
Q
12
47
L
H77C(1a)
A,
R1
V7
40
87
A,
8W
,I
R1
J6/JFH1
13
12
V
TN(1a)
40
8W
J6/JFH1
F8
<2.3
4
12
Infectivity titre [log10(f.f.u. ml–1)]
5
Q
(a)
5
<2.3
HK6a(6a)
J6/JFH1
41
,I
N
N
41
7T
S,
49
N
78
P
QC69(7a)
3
F3
49
S,
F3
02
A1
SA13(5a)
1G
,K
A1
11
R1 02
18
97 1G
R
8G , K
, C 11
24 18
19 R,
R
J6/JFH1
Day 10
68
<2.3
Day 8
22
3
Day 6
4
7T
Infectivity titre [log10(f.f.u. ml–1)]
4
L8
Infectivity titre [log10(f.f.u ml–1)]
Day 3
Fig. 2. Infectivity titres after transfection of Huh7.5 cells with JFH1-based recombinants expressing genotype-specific core–
NS2/NS5A. Infectivity was measured in supernatants from the first 10 days after transfection with RNA transcripts of
recombinants of isolates(genotypes): (a) TN(1a), (b) H77C(1a), J4(1b), S52(3a), (c) SA13(5a), QC69(7a) and (d) HK6a(6a).
The ED43 recombinants with and without Y1644H/E2267G mutations spread slowly to the majority of culture cells (22–27 and
17 days, respectively), and are therefore not shown. For comparison, J6/JFH1 was included in each separate experiment as a
control. Coloured boxes indicate the core–NS2 and NS5A isolate of the given recombinant. Numbering of mutations is
according to the H77 reference polyprotein (GenBank accession no. AF009606). All recombinants were passaged to naı̈ve
Huh7.5 cells to obtain full ORF sequences (Tables S1–S7). The lower limit of detection in the experiments shown was up to
102.3 f.f.u. ml”1; titres below this level are shown as ,2.3 log10(f.f.u. ml”1). Error bars indicate SEM of triplicate titre
determinations.
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Journal of General Virology 94
Co-localization of HCV core and NS5A of genotypes 1–7
After passage to naı̈ve Huh7.5 cells, infectivity titres above
103 f.f.u. ml21 were observed for all recombinants, except
for J4(1b) and ED43(4a). Viral RNA was extracted from
supernatants with peak HCV infectivity titres and sequenced
to obtain the near full-length genomic sequence, including
the entire ORF. Putative adaptive mutations were identified
in all recombinants, except QC69, that apparently did not
depend on additional adaptation (Tables S1–S7, available
with the online version of this paper).
Generation of cell-culture-adapted core–NS2/
NS5A recombinants by reverse genetic analysis
Selected mutations observed for the core–NS2/NS5A recombinants were tested in reverse genetic studies for H77C(1a),
TN(1a), J4(1b), S52(3a), SA13(5a) and HK6a(6a) (Figs 1 and
2). While the SA13(5a)A1021G,K1118R,R1978G,C2419R mutant immediately produced titres comparable to J6/JFH1 in transfection
cultures, the H77C(1a)V787A,C1185S,Q1247L, TN(1a)I1312V,R1408W,
S52(3a)I787S,K1398Q,C2419R
and
HK6a(6a)F349S,N417T,I2268N
mutants produced titres around 103 f.f.u. ml21, and
J4(1b)F886L,C1185S,Q1496L produced titres around 102.5 f.f.u.
ml21 (Table 1). Since the ED43(4a) core–NS2/NS5A recombinant did not adapt to efficient titre production during three
serial passages to naı̈ve cells, mutations identified in recovered
virus (Table S5) were not tested in reverse genetic studies.
We instead inserted the Y1644H and E2267G substitutions previously shown to adapt a J6/JFH1-based recombinant
with ED43 NS5A (Scheel et al., 2011b). The
ED43(4a)T827A,T977S,Y1644H,E2267G recombinant produced titres
around 102.5 f.f.u. ml21 (Table 1) 22 days after transfection.
After passage of the final genotype 1–7 core–NS2/NS5A
recombinants to naı̈ve Huh7.5 cells, peak HCV RNA titres
were around 107 IU ml21 (Table 1). Peak HCV infectivity
titres were around 104 f.f.u. ml21 for S52(3a), SA13(5a)
and QC69(7a), 103.5 f.f.u. ml21 for H77C(1a) and
HK6a(6a), and below 103 f.f.u. ml21 for TN(1a), J4(1b)
and ED43(4a) (Table 1). No additional mutations were
identified in the recovered viruses by sequencing of the
complete ORF, except for the TN(1a) and ED43(4a)
(Tables S1–S7). Thus, we developed a genotype-specific
panel of core–NS2/NS5A recombinants producing infectious virus, which could be useful for functional studies.
Except for the SA13(5a) and QC69(7a) recombinants, virus
production was lower for these recombinants compared
with previously developed J6/JFH1-based NS5A recombinants (Scheel et al., 2011b), and JFH1-based core–NS2
recombinants (Gottwein et al., 2009; Scheel et al., 2011a).
Validation of labelling efficiency of core and
NS5A proteins from HCV genotypes 1–7 in
infected Huh7.5 cells
Proper evaluation of co-localization of core and NS5A from the
different HCV recombinants relies on comparable efficiency of
protein labelling and signal intensity across genotypes.
Therefore, we initially performed single-staining of core and
NS5A from Huh7.5 cells infected with each core–NS2/
NS5A recombinant to verify reactivity of primary antibodies against different genotypes. Supernatants containing
passaged H77C(1a)V787A,C1185S,Q1247L, TN(1a)I1312V,R1408W,
J4(1b)F886L,C1185S,Q1496L, J6/JFH1, S52(3a)I787S,K1398Q,C2419R,
ED43(4a)T827A,T977S,Y1644H,E2267G, SA13(5a)A1021G,K1118R,R1978G,C2419R,
HK6a(6a)F349S,N417T,I2268N and QC69(7a)L878P viruses (Table
1) were used to infect naı̈ve Huh7.5 cells. Cells were fixed at
48 h post-infection and stained using primary antibodies
Table 1. Characteristics of culture-adapted core–NS2/NS5A HCV recombinants
Core–NS2/NS5A
Engineered mutations*
Genotype Isolate
Peak titresD
Transfection
log10(f.f.u. ml”1)
1a
1b
2a
3a
4a
5a
6a
7a
H77C
TN
J4
J6/JFH1§
S52
ED43
SA13
HK6a
QC69
V787A, C1185S, Q1247L
I1312V, R1408W
F886L, C1185S, Q1496L
I787S, K1398Q, C2419R
T827A, T977S, Y1644H, E2267G
A1021G, K1118R, R1978G, C2419R
F349S, N417T, I2268N
L878P
2.9
2.7
2.5
4.2
3.5
2.6
4.5
3.0
3.4
Pearson’s
coefficientd
Viral passage
log10(f.f.u. ml”1)
3.5
2.5
1.8
5.0
3.8
2.3
3.8
3.3
4.1
log10(IU ml”1)
7.5
6.8
7.3
7.6
7.7
7.2
7.1
7.1
7.7
0.82
0.77
0.73
0.77
0.80
0.79
0.78
0.75
0.76
*Mutations are numbered according to the H77 reference polyprotein (GenBank accession no. AF009606). Sequencing results of virus recovered
from supernatants are shown in Tables S1–S7. Mutations introduced in this study are shown in bold.
DRepresentative infectivity (f.f.u.) and RNA (IU) titres observed in supernatants from transfection and passages to naı̈ve cells are indicated.
dPearson’s coefficients represent mean values calculated using image stacks from three to five infected cells per sample, after image alignment and
three-dimensional (3D) deconvolution.
§J6/JFH1 carries the core–NS2 region from J6CF and the NS5A gene from JFH1. Titre data shown are from Gottwein et al. (2009).
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A. Galli and others
anti-NS5A–mIgG1 (9E10) and anti-core–mIgG2a (1851). In
single-staining, both antibodies had good reactivity against
all genotypes (Fig. S1). However, in double-staining
protocols assessing core/NS5A co-localization, several genotypes showed a markedly reduced labelling of core protein
(data not shown). Three anti-core antibodies (anti-core–
mIgG2a 1851, 1868 and C7-50), binding to different
epitopes, were subsequently used in 1 : 1 : 1 concentration
ratio for double-staining with anti-NS5A, and showed
efficient and consistent labelling across the genotype panel.
Comparison of double-staining with anti-NS5A and either
one or three anti-core antibodies was performed on the J6/
JFH1 and SA13(5a) recombinants (Fig. S2). Since there was
no detectable increase in background using three instead of
one antibody, triple core staining was used in all subsequent
experiments. To verify the specificity of our staining
protocol and exclude the possibility of cross-talk between
To validate our analysis protocol, we initially single-stained
J6/JFH1-infected cells with either anti-NS5A or anti-core
primary antibodies, followed by double-staining with
secondary antibodies conjugated to Alexa Fluor 488 and
Core
Alexa488
Alexa546
Alexa488
NS5A
Alexa546
ED43(4a)
NS5A
Alexa488
Alexa546
Co-localization analysis of genotype 1–7 core and
NS5A proteins in infected Huh7.5 cells
S52(3a)
Control
JFH1(2a)
QC69(7a)
J6/JFH1(2a)
J4(1b)
HK6a(6a)
TN(1a)
SA13(5a)
H77(1a)
Core
Alexa488
Alexa546
channels, cells infected with passaged viruses were fixed at
48 h post-infection, stained with either primary anti-core or
anti-NS5A antibodies, and with both Alexa Fluor 488- and
Alexa Fluor 546-conjugated secondary antibodies. All
samples exhibited highly specific staining, with fluorescence
detected exclusively in the correct channel (Fig. 3). Thus, we
developed an efficient and specific labelling strategy for
double-staining of core and NS5A proteins from the seven
major HCV genotypes.
Fig. 3. Cross-talk controls for single-staining of core or NS5A protein on Huh7.5 cells infected with genotype 1–7 core–NS2/
NS5A recombinants and the original JFH1 virus. Cells fixed at 48 h post-infection were stained with either anti-NS5A–mIgG2a
or three anti-core–mIgG1 antibodies. Secondary staining was performed using both anti-mIgG2a–Alexa Fluor 488 and antimIgG1–Alexa Fluor 546. Panels show maximum intensity projections (MIPs) of two-channel stacks from cells infected with
different genotypes and subtypes. In the headings, core and NS5A indicate the primary antibody used to stain each set of
columns, while Alexa488 and Alexa546 indicate the acquisition channel shown in each column. Images were deconvolved and
enhanced for publication. Control, stained uninfected cells.
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Journal of General Virology 94
Co-localization of HCV core and NS5A of genotypes 1–7
Alexa Fluor 546 fluorophores. Images obtained for doublestained cells showed a near-perfect signal overlap (Fig. 4a),
and Pearson’s coefficients of 0.96 and 0.94 for NS5A and
core protein, respectively, close to the theoretical value of 1
(Fig. 4c). Conversely, cells infected with J6/JFH1 and
processed using anti-NS5A and anti-core antibodies
together, following our standard double-staining protocol,
displayed a partial overlap of NS5A and core signals (Fig.
4b) and a Pearson’s coefficient of 0.77 (Fig. 4c), similar to
what was previously reported for the J6/JFH1 recombinant
Jc1 (Appel et al., 2008). These data indicated that our
image post-processing and analysis protocol correctly
detected and interpreted the proteins fluorescent signals.
[J6/JFH1(2a), SA13(5a)], intermediate infectivity [S52(3a)]
or low infectivity [TN(1a), JFH1(2a)]. Protein and cLD
distribution was assessed at 48 h post-infection using our
optimized protocol, adjusted to accommodate lipid staining with boron-dipyrromethene (BODIPY) (Fig. 5a).
To investigate whether there are differences in the core/NS5A
association among HCV genotypes, we performed colocalization analysis of the two proteins using the novel
core–NS2/NS5A genotype 1–7 recombinants. The original
JFH1 virus (Wakita et al., 2005) was included in the analysis
as a control for cLD-associated distribution of core.
Supernatants containing passaged viruses (Table 1) were
used to infect Huh7.5 cells, which were stained at 48 h postinfection. Analysis of core and NS5A staining showed a
partial overlap of core and NS5A signals in all samples (Fig.
4b), and comparable signal intensities for the different
constructs. Core–NS2/NS5A recombinants showed both
proteins dispersed within the cytoplasm in discrete aggregates, distributed in a reticular manner suggestive of
localization on the ER. Conversely, JFH1 virus displayed
the typical condensed distribution of core protein, indicative
of association with the surface of cLDs. Co-localization
analysis of core and NS5A proteins from core–NS2/NS5A
recombinant viruses produced Pearson’s coefficients between
0.73 and 0.82 (Fig. 4c, Table 1) and showed no significant
difference in core/NS5A co-localization among genotypespecific recombinants (one-way ANOVA test; P50.66). In
contrast, JFH1 showed a significantly lower co-localization
coefficient compared with the core–NS2/NS5A constructs
(one-way ANOVA test; P,0.05). These results indicated that
the core and NS5A proteins from efficient recombinants
representing the seven major HCV genotypes have similar
distributions during the viral life cycle.
To quantify the level of association between core and cLDs,
we evaluated the percentage of threshold core signal that colocalized with threshold cLDs signal (as implemented in the
IMARIS software). This should give a more objective estimate
of core/cLD association compared with the commonly used
count of core-positive cLDs and result in a better
representation of cells with intermediate association levels.
JFH1 showed a significantly higher level of core/cLD colocalization (37.5 %), compared with co-localization levels
produced by the core–NS2/NS5A recombinants (3.7–10.4 %)
(one-way ANOVA test; P,0.001) (Fig. 5c). Genotypespecific recombinants with low [TN(1a)] or intermediate
[S52(3a)] infectivity titres displayed relatively higher colocalization levels compared with high infectivity viruses [J6/
JFH1(2a), SA13(5a)]. However, only TN(1a) and SA13(5a)
were significantly different in the co-localization of core and
cLD signal (one-way ANOVA test; P,0.05). These results
showed that core–NS2/NS5 recombinants representing four
major HCV genotypes have comparable association levels of
core and cLDs overall. However, our data also suggested that
genotype-specific alterations of core/cLD association might
be correlated with differences in viral assembly or release.
Analysis of core and cLD association in Huh7.5
cells infected with core–NS2/NS5A genotype
recombinants
cLDs play an important role in the life cycle of HCV,
functioning as scaffold for the recruitment of core proteins
and initiation of new virion assembly. JFH1 core protein is
known to localize almost exclusively on the surface of cLDs
(Miyanari et al., 2007), whereas core protein from the Jc1
recombinant is distributed mostly on the surface of the ER
(Boson et al., 2011). To assess whether other HCV
genotypes display differences in the core/cLD distribution,
we performed co-localization analysis on cells triplestained for core, NS5A and cLDs. Huh7.5 cells were
infected using recombinant viruses with high infectivity
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Co-localization analysis of core and NS5A proteins from
core–NS2/NS5A recombinant viruses produced Pearson’s
coefficients between 0.68 and 0.76, comparable to our
previous experiments (Fig. 5b). There was no significant
difference among co-localization coefficients obtained
from genotype-specific recombinants or compared with
JFH1, despite the fact that the latter produced a markedly
lower coefficient (one-way ANOVA test; P50.54 and P50.50).
Time-lapse analysis of core/NS5A and core/cLD
co-localization in Huh7.5 cells infected with core–
NS2/NS5A genotype recombinants
All analyses performed so far were conducted on cells fixed
and stained at 48 h after infection. To investigate whether
different HCV genotypes display changes in the distribution of core/NS5A/cLDs at different time points after
infection, we performed a time-lapse experiment on cells
infected with TN(1a), J6/JFH1(2a) and SA13(5a) genotypespecific recombinants. Huh7.5 cells were infected in
triplicate, and fixed at 12, 48 and 72 h post-infection. All
time points were then simultaneously stained for core,
NS5A and cLDs using the three-colour staining protocol
introduced previously. Overall, we could not observe major
differences in protein or cLD distribution among the
analysed genotypes (Fig. 6a). Interestingly, all three core–
NS2/NS5A recombinants showed very condensed signals of
NS5A and especially core protein at 12 h, almost perfectly
overlapping with the cLD signal. Later time points showed
more diffuse signals of both proteins, compatible with ER
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2227
A. Galli and others
(a)
Core
Core
Merged
(c)
NS5A
Merged
0.75
*
0.50
0.25
0.00
H
77
(1
TN a)
(1
a)
J6 J4
/J (1b
FH )
1
JF (2a
H )
1
S5 (2a
2 )
ED (3a
43 )
SA (4
13 a)
H (5a
K6 )
Q a(6
C a)
J6 69
/J
F (7
J6 H1 a)
/J
FH Co
1 re
N
S5
A
J6/JFH1
NS5A
Core/NS5A co-localization
(Pearson’s coefficient)
J6/JFH1
1.00
Core
NS5A
Merged
Core
NS5A
Merged
JFH1(2a)
QC69(7a)
HK6a(6a)
J6/JFH1(2a)
J4(1b)
SA13(5a)
TN(1a)
ED43(4a)
S52(3a)
H77(1a)
(b)
Fig. 4. Co-localization analysis of core and NS5A proteins on Huh7.5 cells infected with genotype 1–7 core–NS2/NS5A HCV
recombinants and the original JFH1 virus. (a) Double-staining controls of core and NS5A proteins were infected using J6/JFH1,
fixed at 48 h post-infection and stained using either anti-core–mIgG1 or anti-NS5A–mIgG2a antibodies. Secondary staining
was performed using either anti-mIgG1 or anti-mIgG2a antibodies, each conjugated to Alexa Fluor 488 (green) and Alexa Fluor
546 (red). (b) Cells infected with core–NS2/NS5A recombinants or JFH1 were fixed at 48 h post-infection and double stained
with anti-NS5A–mIgG2a and anti-core–mIgG1 antibodies. Secondary staining was carried out using antibodies anti-mIgG2a–
Alexa Fluor 488 (green, NS5A) and anti-mIgG1–Alexa Fluor 546 (red, core). Pictures show MIPs of two-channel image stacks
of 40–50 slices each. White squared areas are shown enlarged to enhance details. Images were deconvolved and enhanced
for publication. (c) Distribution of Pearson’s coefficients of core/NS5A co-localization among core–NS2/NS5A recombinants
and the original JFH1 virus. The ‘J6/JFH1 core’ and ‘J6/JFH1 NS5A’ bars represent co-localization controls where a single
protein, either core or NS5A, was stained with two colours. Error bars represent SEM obtained from three to five infected cells
from independent infections. *P,0.05.
2228
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Journal of General Virology 94
Co-localization of HCV core and NS5A of genotypes 1–7
(a)
cLDs
NS5A
Merged
SA13(5a)
S52(3a)
JFH1(2a)
J6/JFH1(2a)
TN(1a)
Core
(c)
*
***
0.75
40
0.50
20
0.25
0.00
Core/cLD co-localization
(% core signal overlap)
60
1.00
2(
3a
)
SA
13
(5
a)
2a
)
S5
)
2a
1(
JF
H
1(
a)
H
TN
(5
13
(1
JF
J6
/
a)
)
3a
SA
2(
2a
)
S5
2a
H
1(
(1
JF
TN
J6
/
JF
H
1(
)
0
a)
Core/NS5A co-localization
(Pearson’s coefficient)
(b)
Fig. 5. Co-localization analysis of core/NS5A/cLDs on Huh7.5 cells infected with JFH1 virus or core–NS2/NS5A HCV
recombinants of genotypes 1a, 2a, 3a and 5a. (a) Infected cells were fixed at 48 h post-infection and double stained with anti-NS5A–
mIgG2a and anti-core–mIgG1 antibodies for 24 h at 4 6C. Secondary staining was carried out using BODIPY 505/515 (green,
cLDs) and antibodies anti-mIgG2a–Alexa Fluor 633 (blue, NS5A) and anti-mIgG1–Alexa Fluor 546 (red, core) for 2 h at room
temperature. Pictures show MIPs of representative three-channel image stacks, made up of 50–60 slices each. White squared areas
are shown enlarged to enhance details. Images were deconvolved and enhanced for publication. (b) Distribution of Pearson’s
coefficients of core/NS5A co-localization among genotype-specific recombinants and JFH1. Error bars represent SD obtained from
four infected cells from independent infections. (c) Amount of core protein co-localized with cLDs among genotype-specific
recombinants and JFH1. Values are calculated as the percentage of thresholded core signal that co-localizes with thresholded cLD
signal. Error bars represent SD obtained from four to six infected cells from independent infections. *P,0.05; ***P,0.001.
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2229
(a)
48 h
72 h
SA13(5a)
TN(1a)
J6/JFH1(2a)
12 h
Core/cLD co-localization Core/cLD co-localization Core/NS5A co-localization
(Pearson’s coefficient)
(% core signal overlap) (% core signal overlap)
A. Galli and others
(b)
1.00
*
*
*
0.75
12 h
48 h
72 h
0.50
0.25
0.00
100
TN(1a) J6/JFH1(2a) SA13(5a)
***
***
***
75
(c)
12 h
48 h
72 h
50
0.25
0
TN(1a) J6/JFH1(2a) SA13(5a)
100
***
75
(d)
TN(1a)
J6/JFH1(2a)
SA13(5a)
50
25
*
0
12 h
48 h
72 h
Fig. 6. Time-lapse analysis of core/NS5A/cLD co-localization in Huh7.5 cells infected with core–NS2/NS5A HCV
recombinants of genotypes 1a, 2a and 5a. (a) Infected cells were fixed at 12, 48 or 72 h post-infection and double-stained
with anti-NS5A–mIgG2a and anti-core–mIgG1 antibodies for 24 h at 4 6C. Secondary staining was carried out using BODIPY
505/515 (green, cLDs) and antibodies anti-mIgG2a–Alexa Fluor 633 (blue, NS5A) and anti-mIgG1–Alexa Fluor 546 (red, core)
for 2 h at room temperature. Pictures show merged MIPs of representative three-channel image stacks from each time point,
made up of 50–60 slices each. White squared areas are shown enlarged to enhance details. Images were deconvolved and
enhanced for publication. (b) Distribution of Pearson’s coefficients of core/NS5A co-localization for genotype-specific
recombinants at the indicated time points post-infection. Error bars represent SD obtained from four to eight infected cells from
independent infections. (c, d) Variation of core/cLD association over time for each genotype-specific recombinant (c) and
comparison of core/cLD association among different genotypes over time (d). Values are calculated as the percentage of
thresholded core signal that co-localizes with thresholded cLD signal. Error bars represent SD obtained from four to eight
infected cells from independent infections. *P,0.05; ***P,0.001.
association. Despite a general increase in the size of cLDs,
the 48 and 72 h time points did not display important
differences in signal distribution, either within the same
genotype or among genotypes. Analysis of Pearson’s
coefficients for core/NS5A co-localization showed no
significant differences among genotype-specific constructs
at any time point (two-way ANOVA test; P50.32), whereas
all three recombinants showed a significant difference
between 12 and 72 h (two-way ANOVA test; P,0.001)
(Fig. 6b). Conversely, evaluation of core/cLD association
indicated that SA13(5a) had significantly less core localized
on cLDs than TN(1a) at 12 and 48 h post-infection (two-way
ANOVA test; P,0.001), whereas the difference became nonsignificant at 72 h (Fig. 6d). All three genotypes, however,
displayed significantly more core protein localized on cLDs at
12 h (two-way ANOVA test; P,0.001) (Fig. 6c). Taken
together, these data indicated that in the early stage of the
viral life cycle new particle assembly of core–NS2/NS5A
recombinant viruses is restricted to the surface of cLDs, while
2230
virion assembly is later transferred to the ER surface. Our
results are also consistent with a correlation between
genotype-specific levels core/cLD association and efficiency
of particle assembly/release, as suggested by the comparison
between SA13(5a) and TN(1a) recombinants.
DISCUSSION
In order to be able to study the genotype specific interaction
of the NS5A protein with the structural proteins of HCV, we
developed infectious genotype 1–7 core–NS2/NS5A recombinants. In general, these JFH1-based chimaeras did not
produce as high infectivity titres as previously observed for
core–NS2 or NS5A recombinants, with the exception of the
SA13(5a) and QC69(7a) recombinants (Gottwein et al., 2009;
Scheel et al., 2011b). Differences for peak infectivity titres
observed for the core–NS2/NS5A recombinants reflected
those previously observed for core–NS2 recombinants, where
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Journal of General Virology 94
Co-localization of HCV core and NS5A of genotypes 1–7
S52(3a), SA13(5a) and QC69(7a) were the most efficient
(Gottwein et al., 2009). Apparently, adding a genotypespecific NS5A gene caused a general fitness reduction. While
J6/JFH1-based NS5A recombinants can be of particular
advantage for specific inhibitor and replication studies, since
only the NS5A gene differs (Scheel et al., 2011b, 2012), the
novel core–NS2/NS5A recombinants will be useful for NS5A
interaction studies as shown here and possibly for the
development of full-length genotype-specific culture systems.
In addition, these systems could be useful to assess the efficacy
of novel combination therapies involving NS5A and p7
inhibitors or NS5A inhibitors combined with neutralizing
antibodies.
Most of the recombinants relied on adaptation for efficient
growth, in analogy to what has been reported for intergenotypic JFH1-based core–NS2 and J6/JFH1-based NS5A
recombinants (Gottwein et al., 2007, 2009; Jensen et al.,
2008; Kaul et al., 2007; Scheel et al., 2008, 2011a, b; Yi et al.,
2007). The R1978G mutation conferring adaptation to
SA13(5a) was observed previously for the J6/JFH1-based
NS5A SA13(5a) recombinant, and was demonstrated to
increase the amount of processed NS5A (Scheel et al.,
2011b). The S52(3a) and SA13(5a) recombinants both
acquired C2419R, located within the cleavage site at the Cterminus of NS5A. The neighbouring mutation V2418L, also
in the NS5A/NS5B cleavage site, was shown to slow down
cleavage kinetics, thereby reducing the dependence on
cyclophilin A (Kaul et al., 2009), and it is possible that
C2419R has a similar function. Adaptive mutations in NS5A
domain II improved growth of the ED43(4a) and HK6a(6a)
core–NS2/NS5A recombinants; the HK6a(6a) core–NS2/
NS5A recombinant carried the same mutation observed in
the J6/JFH1-based NS5A HK6a(6a) recombinant (Scheel
et al., 2011b). Such mutations could mediate cross-genotypic
interactions with other HCV proteins interacting with NS5A
(Jirasko et al., 2010; Ma et al., 2011). The H77C(1a) and
J4(1b) core–NS2/NS5A recombinants both acquired the
C1185S mutation close to the active site of the NS3/4A
protease (Kim et al., 1996; Love et al., 1996), possibly
mediating NS5A cleavage compatibility. Furthermore, mutation of the adjacent residue 1187 was shown to influence
NS5A phosphorylation (Lindenbach et al., 2007). I1312V
improved growth kinetics for the TN(1a) core–NS2/NS5A
recombinant. This mutation has been shown to adapt JFH1
and core–NS2 recombinants of genotypes 1a, 1b, 3a and 5a,
suggesting a more general effect in increasing viral fitness in
cell culture (Gottwein et al., 2009; Jensen et al., 2008; Kaul et
al., 2007; Scheel et al., 2008, 2011a).
Several studies have previously investigated co-localization
of different HCV proteins and host cell components; most,
if not all, of these studies have, however, limited their
analyses to genotype 2a culture systems (Appel et al., 2008;
Camus et al., 2013; Herker et al., 2010; Jirasko et al., 2010;
Masaki et al., 2008; Miyanari et al., 2007; Shavinskaya et al.,
2007). In particular, the core and NS5A proteins have been
shown to co-localize extensively in cells infected with
genotype 2a viruses, and their interaction was confirmed to
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be essential for production of infectious particles using
biochemical assays (Appel et al., 2008; Masaki et al., 2008).
In order to analyse the distribution of core and NS5A in
genotypes different from 2a, we optimized a protocol for
efficiently and selectively staining core and NS5A of
genotypes 1–7. Despite the low variability of the core
protein among HCV genotypes, three combined anti-core
antibodies were necessary to achieve comparable staining
across all recombinants. The epitope for the 9E10 NS5A
antibody used here overlaps with aa 414–428, which is
100 % conserved among the isolates analysed here (Scheel
et al., 2012). This probably explains why this antibody
efficiently reacted to all isolates in the current study, but
not to J6 NS5A, which has a single amino acid change in
this region (Scheel et al., 2012). Analysis of the distribution
of core and NS5A signal intensities permitted the
quantification of protein co-localization and the comparison of core/NS5A distribution among different genotypes.
Core and NS5A proteins co-localized to a similar extent in
all recombinants, showing no significant differences
between proteins of different origin. This finding indicates
that the differences in viability among genotype-specific
recombinants were not caused by differences in the core/
NS5A association. This in turn suggests that, in spite of
high sequence variation among the prototype isolates of
genotypes 1–7, the interaction between core and NS5A is
conserved and seems to be essential for HCV. Interestingly,
infection with JFH1 virus resulted in significantly lower
core/NS5A co-localization coefficients compared with
genotype-specific core–NS2/NS5A recombinants. Such
difference can be explained considering that while JFH1
core is mostly associated with cLDs, JFH1 NS5A is largely
localized on the ER, thus resulting in reduced correlation of
the two signals (Boson et al., 2011; Miyanari et al., 2007).
The marked difference in core/NS5A co-localization between
JFH1 and genotype-specific recombinants also indirectly
suggested that the proteins from the novel recombinants
were mostly ER-associated, rather than cLD-associated.
Moreover, the core and NS5A proteins were highly colocalized and distributed within the cytoplasm in a reticular
framework, compatible with association to the ER. We
previously demonstrated co-localization of cLDs and core
signals for the core–NS2 recombinants of genotypes 1–7 that
were used to generate the core–NS2/NS5A recombinants
(Gottwein et al., 2009). In that report, 5–15 % of core protein
co-localized with cLDs, with slight variations between
genotypes. However, we did not observe a correlation
between variations in core/cLD co-localization and virus
production. Here, we analysed core/NS5A/cLD distribution
in cells infected with four core–NS2/NS5A genotype-specific
recombinants and with the original JFH1 virus. Core/NS5A
co-localization coefficients obtained from triple staining was
comparable to what we observed previously, strengthening
our conclusion that different HCV genotypes have a similar
core/NS5A distribution within the cytoplasm. In addition,
we found that 3–10 % of core protein from the novel
recombinants co-localized with cLDs, in line with what we
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2231
A. Galli and others
had observed in our previous study. These figures were
significantly different from the core/cLD co-localization
obtained from JFH1 virus (approximately 40 %), supporting
our conclusion that proteins from the core–NS2/NS5A
recombinants are mostly ER associated. Interestingly, there
seemed to be an inverse correlation between the level of core/
cLD association and the infectivity titres of our novel
recombinants, as had been shown for JFH1 (Appel et al.,
2008; Shavinskaya et al., 2007). Although the measured
differences were relatively small, the two recombinants with
the highest (SA13) and lowest (TN) infectivity titres
produced significantly different levels of core/cLD colocalization. Since reduced accumulation of core and NS5A
proteins on cLDs is indicative of a more efficient assembly of
viral particles, it is possible that variations in core/NS5A/cLD
association could confer genotype-specific differences affecting particle assembly or release.
Finally, to further investigate the differences in the
dynamics of particle assembly of different HCV genotypes,
we performed a time-lapse analysis of core/NS5A/cLD colocalization using genotype-specific recombinants TN(1a),
J6/JFH1(2a) and SA13(5a). All three viruses produced
comparable and increasing levels of core/NS5A colocalization over time, supporting our previous finding
that the protein distribution is independent of the infecting
genotype. Interestingly, core protein from all three
genotype-specific recombinants was highly associated
(.50 %) with cLDs at 12 h post-infection, whereas the
association decreased to ,10 % at later time points. These
data support the hypothesis that the cLDs might serve as
initial storage areas for the accumulation of core before the
actual assembly of viral particles is initiated and transferred
to the surface of the ER. Furthermore, the low-titre TN(1a)
recombinant showed the highest levels of core/cLD
association at all time points, although the differences
were significant only compared with SA13(5a) at 12 and
48 h. These data further support our hypothesis that the
presence of genotype-specific differences in the dynamics
of core/cLD association might affect particle assembly/
release and in turn viral production and infectivity.
In summary, we developed a panel of replicationcompetent HCV recombinants carrying core–NS2 and
NS5A regions of genotypes 1–7, permitting studies of the
interaction of NS5A with HCV structural proteins, p7 and
NS2 for all major genotypes. Moreover, we optimized a
sensitive assay capable of detecting core and NS5A proteins
from seven different genotypes and quantitatively determined their cellular co-localization. We found that core
and NS5A co-localized extensively in all analysed genotypes, and there was no significant difference in protein colocalization among genotypes. In addition, we found that
core and NS5A had a high degree of association with cLDs
at 12 h post-infection but became mostly ER associated at
later stages. Finally, we found that different genotypes
showed varying levels of core/cLD co-localization, with a
possible effect on viral assembly/release. These novel
systems will be useful in functional studies analysing
2232
interaction between core, NS5A and other viral proteins
involved in virion production, such as NS2 and p7, across
HCV genotypes.
METHODS
HCV sources and construction of recombinants. As a backbone
for the novel JFH1-based core–NS2/NS5A genotype recombinants we
used the core–NS2 recombinants H77C/JFH1V787A,Q1247L (Scheel
et al., 2008), TN/JFH1R1408W (Scheel et al., 2011a), J4/JFH1F886L,Q1496L
(Gottwein et al., 2009), S52/JFH1I787S,K1398Q (Gottwein et al., 2009),
ED43/JFH1T827A,T977S (Scheel et al., 2008), SA13/JFH1A1021G,K1118R
(Jensen et al., 2008), HK6a/JFH1F349S,N417T (Gottwein et al., 2009)
and QC69/JFH1L878P (Gottwein et al., 2009). HCV NS5A consensus
sequences of the same prototype isolates (Bukh et al., 2010; Sakai
et al., 2007) were obtained as described previously (Scheel et al.,
2011b). The J6/JFH1 recombinant has been described previously
(Lindenbach et al., 2005). The complete NS5A gene was inserted into
the HCV core–NS2 recombinants by three-piece fusion-PCR,
restriction endonuclease digest and standard cloning procedures.
Mutations in adapted core–NS2/NS5A recombinants were introduced
by site-directed mutagenesis. The complete HCV sequence of all final
plasmid preparations was confirmed (Macrogen).
Cell culture, transfections and infections. Huh7.5 human
hepatoma cells (a gift from C. M. Rice, Rockefeller University, NY,
USA), were handled as described previously (Gottwein et al., 2007).
For transfection, RNA was in vitro transcribed using T7 RNA
polymerase (Promega) on 10 mg DNA plasmid linearized with XbaI
(NEB) and treated with mung bean nuclease (NEB), following the
manufacturers’ recommendations. Transfection was performed using
2.5 mg RNA with 5 ml Lipofectamine 2000 (Invitrogen) in Opti-MEM
(Invitrogen) on 46105 cells plated the day before in six-well plates
(Nunc). For infection, culture-derived sterile-filtered supernatants,
stored at 280 uC, were inoculated onto 46105 cells plated the day
before in six-well plates. Infections for co-localization studies were
performed at a low m.o.i. (0.01–0.05) on 26104 cells plated the day
before in eight-well slides (Nunc).
Evaluation of infection and viral titres. In core–NS2/NS5A
recombinant development cultures, infected cells were monitored by
immunofluorescence for HCV core. Primary antibody was mouse antiHCV core protein mAb (B2; Anogen) and secondary antibody was Alexa
Fluor 594 goat anti-mouse IgG (H+L) (Invitrogen). HCV RNA titres
were determined by a TaqMan real-time PCR assay on the 59 UTR
(Gottwein et al., 2007). Infectivity titres were determined by inoculating
100 ml of triplicate sample dilutions on 66103 cells per well plated out
the day before on poly-D-lysine-coated 96-well plates (Nunc). At 48 h
after infection, cells were fixed and immunostained for HCV protein
expression using a previously established protocol (Lindenbach et al.,
2005). Primary antibody was HCV NS3 antibody (H23; Abcam),
recognizing the NS3 helicase domain. Secondary antibody was ECL antimouse IgG HRP-linked whole antibody (GE Healthcare Amersham).
Staining was developed using DAB substrate kit (Dako). The number of
f.f.u. was determined on an ImmunoSpot Series 5 UV Analyser (CTL
Europe) with customized software (Gottwein et al., 2010) or manually by
counting under a microscope. For automatic counting, the mean f.f.u.
count of six negative control wells was subtracted from f.f.u. counts in
experimental wells. Lower limit of quantification was set to 3 SD above
the negative mean, corresponding to 102–102.3 f.f.u. ml21. Counts of up
to 200 f.f.u. per well were in the linear range of a test dilution series and
were comparable to manual counts.
Sequence determination of culture-derived HCV. RNA extrac-
tion, reverse transcription-PCR and direct sequence analysis were as
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Journal of General Virology 94
Co-localization of HCV core and NS5A of genotypes 1–7
described previously (Gottwein et al., 2007, 2009; Jensen et al., 2008;
Scheel et al., 2008). Primers specific for the NS5A region were as given
by Scheel et al. (2011b). All sequencing was performed by Macrogen.
Sequence analysis was performed with SEQUENCHER (Gene Codes
Corporation). HCV reference sequences were retrieved from the
European HCV database and the Los Alamos HCV sequence
database.
Immunostaining of HCV proteins for cellular co-localization
studies. Cells were washed three times with PBS 48 h post-infection,
fixed with 4 % paraformaldehyde and 0.02 % glutaraldehyde for 10 min
at room temperature, and washed three times with PBS.
Permeabilization, blocking and quenching were performed in a single
step by incubating fixed cells with PBS containing 0.5 % Saponin, 10 %
goat serum, 3 % BSA, 0.02 % skimmed milk and 0.1 M glycine for
30 min at room temperature. After washing with PBS, primary
antibodies diluted in PBS containing 3 % BSA and 0.02 % skimmed
milk were added to the cells and incubated at 4 uC for 24 h. NS5A was
detected using the clone 9E10 mouse IgG2a mAb (kindly provided by C.
M. Rice) at 1 : 200 dilution; core protein was detected using either the
single mouse IgG2a mAb clone 1851 Monotope (Virostat) at 1 : 100
dilution or a mix of three mouse IgG1 mAbs at a 1 : 100 dilution: clones
1851 and 1868 Monotope (Virostat) and clone C7-50 (Enzo Life
Sciences). Cells were then washed three times with PBS and incubated
for 2 h at room temperature with secondary antibodies diluted 1 : 1000
in PBS containing Hoechst 33258 (Sigma). Single-staining experiments
were conducted using Alexa Fluor 488-conjugated goat anti-mIgG
antibody (Invitrogen); double-staining was performed using Alexa
Fluor 488-conjugated goat anti-mIgG2a and Alexa Fluor 546-conjugated goat anti-mIgG1 antibodies; triple-staining experiments were
carried out using BODIPY 505/515 at a 1 : 500 dilution, and Alexa Fluor
546-conjugated goat anti-mIgG1 and Alexa Fluor 633-conjugated goat
anti-mIgG2a. Dual staining of NS5A or core alone was carried out using
Alexa Fluor 488 and Alexa Fluor 546 conjugated to goat anti-mIgG1 (for
core) or goat anti-mIgG2a (for NS5A). Finally, cells were washed with
PBS and mounted using Fluoromount-G (Southern Biotech).
Image acquisition and analysis. Images were acquired avoiding
pixel saturation using a Leica TCS SP2 laser scanning microscope,
equipped with a Plan-Apochromat 663 objective (numerical aperture
1.4). To ensure collection of all sample information during acquisition,
images were recorded following or exceeding Nyquist sampling
parameters, as calculated using the online Nyquist Calculator tool
(http://www.svi.nl/NyquistCalculator). Thus, acquisition was performed using 66 electronic zoom with 102461024 pixel resolution,
resulting in a voxel size of 39 nm for the x and y dimensions and
122 nm for the z dimension. To avoid the occurrence of fluorescence
cross-talk, images from each channel were acquired sequentially. For
3D deconvolution, image stacks of 40–50 slices per channel were
acquired for each cell. AUTOQUANT software (MediaCybernetics) was
used for image alignment and 3D deconvolution. Signal co-localization
was evaluated by Pearson’s coefficient using IMARIS software (Bitplane).
This coefficient represents the ratio between the channel covariance
and the product of their SDs, quantifying the linear correlation of the
two channels independent of their intensities. Localization of core on
cLDs was evaluated by measuring the percentage of total core signal
intensity above a set threshold that co-localized with signal from cLDs
above its threshold, using IMARIS software. This value gives a measure of
the co-occurrence of the two signals taking into account their
intensities, but not their linear correlation. Statistical analyses were
performed using Graphpad PRISM software.
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ACKNOWLEDGEMENTS
We thank L. Ghanem and A.-L. Sørensen for general laboratory
assistance. J. O. Nielsen and O. Andersen (Copenhagen University
http://vir.sgmjournals.org
Hospital, Hvidovre) provided valuable support, and C. M. Rice
(Rockefeller University, NY, USA) provided reagents. A. G. was
supported by Postdoctoral grants from the Lundbeck Foundation and
Marie Curie International Reintegration Grant; T. K. H. S. and J. P. R.
were supported by PhD stipends from Faculty of Health Sciences,
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T. K. H. S. was in addition supported by a Sapere Aude Research
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from Lundbeck Foundation (J. M. G. and J. B.), the Danish Cancer
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