JVI Accepts, published online ahead of print on 25 June 2014 J. Virol. doi:10.1128/JVI.00586-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Title
2
Influenza A virus hemagglutinin and neuraminidase mutually accelerate their
3
apical targeting through clustering of lipid rafts.
4 5
Running title
6
Apical targeting of HA and NA via lipid raft
7 8
Takashi Ohkura,a Fumitaka Momose,a Reiko Ichikawa,a Kaoru Takeuchi,b and Yuko
9
Morikawaa
10
a
11
University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
12
b
13
of Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan
Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato Department of Environmental Microbiology, Division of Biomedical Science, Faculty
14 15
Corresponding author:
16
Yuko Morikawa
17
Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato
18
University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan.
19
Phone:
20
[email protected]
+81-3-5791-6129;
Fax:
+81-3-5791-6268;
E-mail:
21
Abstract
22 23
In polarized epithelial cells, influenza A virus hemagglutinin (HA) and
24
neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical
25
plasma membrane for viral assembly and budding. Previous studies have indicated that
26
the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA were
27
required for association with lipid rafts, but the raft dependencies of their apical
28
targeting are controversial. Here, we show that coexpression of HA with NA accelerated
29
their apical targeting through accumulation in lipid rafts. HA was targeted to the apical
30
even when expressed alone but the kinetics was much slower than that of HA in infected
31
cells. Coexpression experiments revealed that apical targeting of HA and NA was
32
accelerated by their coexpression. The apical targeting of HA was also accelerated by
33
coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and
34
the deletion of the CT in HA and NA that reduced their association with lipid rafts
35
abolished the acceleration of their apical transport, indicating that the lipid raft
36
association is essential for efficient apical trafficking of HA and NA. An in situ
37
proximity ligation assay (PLA) revealed that HA and NA were accumulated and
38
clustered in the cytoplasmic compartments, only when both were associated with lipid
39
rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings.
40
We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism
41
of the accelerated apical transport of HA and NA via clustering of lipid rafts.
42
43
Importance
44 45
Lipid rafts serve as sites for viral entry, particle assembly, and budding, leading
46
to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma
47
membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase
48
(NA) of influenza virus, key players for particle assembly, contain determinants for
49
apical sorting and lipid raft association. However, it remains to be elucidated how lipid
50
rafts contribute to the apical trafficking and budding. We investigated the relation of
51
lipid raft association of HA and NA to the efficiency of apical trafficking. We show that
52
coexpression of HA and NA induces their accumulation in lipid rafts and accelerates
53
their apical targeting and suggest that the accelerated apical transport likely occurs by
54
clustering of lipid rafts at the TGN. This finding provides the first evidence that two
55
different raft-associated viral proteins induce lipid raft clustering, thereby accelerating
56
apical trafficking of the viral proteins.
57
Introduction
58 59
Influenza virus is an enveloped, negative-stranded, segmented RNA virus
60
belonging to the Orthomixoviridae family. The virion consists of three integral
61
membrane proteins, hemagglutinin (HA), neuraminidase (NA), and ion channel protein
62
M2. A layer of matrix protein M1 is present underneath the lipid envelope and encases
63
viral ribonucleoprotein (vRNP) complexes. The influenza virus buds from the apical
64
plasma membrane (PM), which is divided by tight junctions in polarized epithelial cells
65
(1). It is considered that all viral components are targeted to the apical PM, where
66
particle budding occurs. HA, NA, and M2 are synthesized at the endoplasmic reticulum
67
(ER) and are transported to the apical PM through the trans-Golgi network (TGN). The
68
apical sorting signals were identified in the transmembrane domain (TMD) of both HA
69
and NA (2, 3). Many studies indicate that during the apical trafficking, HA and NA are
70
associated with lipid raft microdomains, which are enriched in cholesterol and
71
sphingolipids (3, 4), whereas M2 is excluded from these domains (5, 6). Several studies
72
also indicate that the TMD and the cytoplasmic tail (CT) of HA and NA are important
73
for their association with lipid rafts (3, 5, 7). It has been shown that, in the case of HA,
74
palmitoylation at three conserved cysteines in the TMD-CT region is required for
75
association with lipid rafts (8). A very recent study suggested that M2 was a key player
76
in influenza virus particle budding, which is endosomal protein sorting complex
77
required for transport (ESCRT)-independent (9).
78
Lipid rafts are thought to function as platforms for selective concentration of
79
raft-associated proteins to promote protein-protein interactions for their functions (10).
80
Lipid rafts have also been shown to play pivotal roles in apical trafficking in polarized
81
cells (11) and in signal transduction pathways, such as Ras signaling (12) and PIP2
82
signaling (13). It has been suggested that for influenza virus HA and NA, the
83
association with lipid rafts constitutes a part of the machinery necessary for apical
84
trafficking in polarized cells (14, 15). Previous studies have indicated that disruption of
85
lipid rafts by treatment with methyl-β-cyclodextrin (MβCD) and lovastatin delays the
86
TGN-to-apical PM trafficking of HA and missorts to the basolateral membrane, whereas
87
vesicular stomatitis virus G protein, a nonraft-associated basolateral marker, remained
88
unaffected (16), suggesting that raft association is required for apical transport of
89
proteins. However, a number of studies have indicated that some mutations in the HA
90
TMD and CT did not impair apical targeting of HA irrespective of whether or not they
91
caused significant reductions in the raft association (5, 7), suggesting that raft
92
association is not essential for apical transport of HA.
93
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are not
94
integral membrane proteins but are associated with lipid rafts through the GPI moieties.
95
In this group of proteins, the raft association is not a determinant for apical sorting
96
because both apical and basolateral GPI-APs are associated with lipid rafts (17).
97
Interestingly, only apical, but not basolateral, GPI-APs form oligomer complexes when
98
they are associated with lipid rafts. When oligomerization of GPI-APs was impaired by
99
mutations, the GPI-APs were missorted to the basolateral PM domain (17). A recent
100
model has suggested that the oligomerization of GPI-APs promotes their stabilization in
101
lipid rafts, leading to their incorporation into apical transport vesicles (10).
102
Influenza virus budding and release require the assembly of viral components,
103
which occurs either during their trafficking to the apical PM or at the stage of particle
104
budding (6, 18, 19). During the trafficking, all viral components, HA, NA, M2, M1, and
105
vRNP, either individually or in their complex forms, are targeted to the apical PM and
106
form a higher order of complex (7, 19, 20). Although the apical sorting determinants for
107
individual viral components have been relatively well studied (2, 3), the molecular
108
mechanisms and kinetics involved in their apical targeting have not been elucidated.
109
In this study, we focused on the kinetics of apical targeting of influenza virus
110
envelope proteins in polarized MDCK cells. We found that HA and NA or M1, but not
111
M2, mutually accelerated their apical PM targeting. Using the TMD-CT mutants of HA
112
and NA, we show that the association of HA and NA with lipid rafts is necessary for the
113
acceleration of their apical PM targeting. Our data indicate that HA and NA come into
114
close proximity (clustering) in lipid rafts upon coexpression. Our data also show that
115
clustering of lipid rafts upon coexpression of HA and NA, suggesting a possible
116
mechanism of accelerated apical transport of HA and NA via the clustering of lipid rafts.
117
Materials and methods
118 119
Viruses and plasmids The H141Y and E142Q mutations were introduced into the HA gene of the
120 121
influenza A/Puerto Rico/8/34 (PR8) virus by inverted PCR. This PR8 derivative was
122
referred to as the wild type (wt) strain in the present study. The authentic PR8 strain was
123
used as the parental virus. The wt and mutant PR8 viruses were generated by reverse
124
genetics system with PolI plasmids (pHH21) and protein expression plasmids, as
125
described previously (21). Briefly, 293T cells were transfected with PolI plasmids for
126
synthesis of each viral RNA segment and protein expression plasmids for PB1, PB2, PA,
127
NP, HA and NA. At 6 h post-transfection (hpt), the cell medium was replaced with
128
Opti-MEM I (Gibco) supplemented with 5 μg/ml acetyl trypsin and 0.3% bovine serum
129
albumin (BSA), and the cells were incubated for 2 or 3 days. Recovered viruses were
130
grown in Madin-Darby canine kidney (MDCK) cells stably expressing HA
131
(MDCK-HA) (the kind gift of Dr. Takizawa from the Institute of Microbial Chemistry,
132
Japan).
133
The HA and NA constructs with deletion of the CT (ΔCT-HA and ΔCT-NA) for
134
recovery of recombinant viruses have essentially been described elsewhere (22, 23). For
135
ΔCT-HA, three consecutive stop codons were placed at the end of the TMD-coding
136
sequence and the downstream nucleotide sequence was left intact. For ΔCT-NA, the
137
authentic start codon was mutated and a new start codon was created at the beginning of
138
the TMD-coding sequence. The constructs were cloned into pHH21. The HA and NA
139
constructs with alanine substitutions in the CT (HA557-559, HA560-563, HA564-566,
140
NA2-3, NA4-6, and NA2-6) and TMD (HA533-535, HA550-552, NA7-10, and
141
NA31-35), and those with cysteine-to-serine substitutions at three palmitoylation sites
142
(HA-SSS), were generated by overlapping PCR and were cloned into pHH21. The
143
viruses with similar alanine substitutions have been described previously (3, 5, 8).
144
The open reading frames of the wt-HA, NA, M1, and M2 genes were cloned
145
into the eukaryotic expression plasmid pCAGGS, respectively. The open reading frames
146
of the HA and NA constructs with deletion of the CT and those with the alanine
147
substitutions were similarly cloned into pCAGGS, respectively. The cDNAs encoding
148
CD59, the 75 kDa neurotrophin receptor (p75), and their GFP-tagged versions
149
(GFP-CD59 and p75-GFP) were also cloned into pCAGGS.
150 151
Cell culture, infection, and DNA transfection
152
MDCK and 293T cells were maintained in Dulbecco’s modified Eagle’s
153
medium (Sigma) supplemented with 10% fetal bovine serum. MDCK cells were seeded
154
into 12-well plates (1×106 cells/well) and were polarized by 12 h-incubation. The
155
polarized MDCK cells were used throughout this study. The MDCK cells were infected
156
with the wt virus at a multiplicity of infection (MOI) of 0.1 or 1 for 1 h. Transfection of
157
DNA was carried out using Lipofectamine LTX (Invitrogen). To synchronize protein
158
expression from plasmids, DNA-Lipofectamine complexes were sedimented by plate
159
centrifugation at 250 × g for 5 min. In some experiments, MDCK cells were transfected
160
with DNA and subsequently superinfected with the authentic PR8 virus.
161 162
Virus growth and plaque assay
163
MDCK cells were inoculated with virus at an MOI of 0.1 in Opti-MEM I
164
supplemented with 0.3% BSA for 1 h at 37°C. After being washed, the cells were
165
incubated in Opti-MEM I supplemented with 5 μg/ml acetyl trypsin and 0.3% BSA. The
166
culture medium was harvested at various time points and was subjected to plaque assay
167
on MDCK or MDCK-HA cells.
168 169
Cholesterol depletion
170
For the inhibition of cholesterol synthesis, 293T and MDCK cells were
171
pre-treated with 8 μM lovastatin (Merck) for 12 h. After transfection, the cells were
172
incubated in the presence of 8 μM lovastatin for 12 or 24 h. The cells were further
173
treated with 5 or 10 mM methyl-β-cyclodextrin (MβCD) (Sigma) in the presence of 8
174
μM lovastatin for 1 h.
175 176
Indirect immunofluorescence assay
177
Polarized MDCK cells were grown on coverslips in 12-well plates and were
178
either infected with virus or transfected with protein expression plasmids. The cells
179
were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton
180
X-100 (TX-100). Following blocking, the cells were incubated with primary antibodies
181
(Abs) and subsequently with secondary Abs conjugated with Alexa Fluor 488, 568, or
182
647 (Molecular Probes). The following Abs were used as primary Abs: mouse anti-HA
183
mAb12-1G6 (24), mouse anti-HA Ab (Takara), rabbit anti-NA Ab (Sino Biological),
184
sheep anti-NA Ab (R&D Systems), rabbit anti-M1 polyclonal Ab (25), mouse anti-M2
185
Ab (Santa Cruz), mouse anti-NP mAb61A5 (26), mouse anti-CD59 Ab (Abcam), rabbit
186
anti-TGN46 Ab (Abcam), rabbit anti-ZO-3 Ab (Invitrogen), and rabbit anti-caveolin-1
187
Ab (Santa Cruz). For costaining with HA and M2 or vRNP, anti-HA mAb12-1G6 was
188
pre-labeled by using the Zenon Alexa Fluor 488 mouse IgG1 labeling kit (Invitrogen),
189
and the mouse anti-HA (Takara), anti-M2, and anti-NP Abs were similarly pre-labeled
190
with Alexa Fluor 568, as described previously (24). Nuclear staining was carried out
191
with TO-PRO-3 (Molecular Probes) or DAPI (Molecular Probes). The cells were
192
observed with a laser scanning confocal microscope (TCS-SP5II AOBS; Leica).
193
Confocal images were collected at 0.5-μm intervals along the Z-axis. Reconstitution of
194
an X-Z plane was processed using ImageJ software (27). Fifty antigen-positive cells
195
were observed in each experiment and patterns of antigen distribution were analyzed.
196
For quantitative analysis of HA and NA expression, polarized MDCK cells on
197
coverslips in 12-well plates (1×106 cells/well) were singly transfected with an HA
198
expression plasmid or cotransfected with the HA and an NA expression plasmids. After
199
fixation with 4% PFA, the cells were incubated with mouse anti-HA Ab (Takara) (for
200
HA on the apical PM). The cells were re-fixed with 4% PFA and were permeabilized
201
with 0.5% TX-100. The cells were incubated with rabbit anti-NA Ab (Sino Biological)
202
(for total NA) and subsequently with anti-mouse IgG conjugated with Alexa Fluor 647
203
and anti-rabbit IgG conjugated with Alexa Fluor 568. For total HA, the cells were
204
re-fixed with 4% PFA and were blocked with nonspecific mouse Ig. The cells were
205
finally incubated with anti-HA mAb12-1G6 pre-labeled by using the Zenon Alexa Fluor
206
488 mouse IgG labeling kit (Invitrogen). Nuclear staining was carried out with DAPI
207
(Molecular Probes). Confocal images were collected at 0.5-μm intervals along the
208
Z-axis. In each cell, the sum of intensity values of a Z-stack was calculated as a
209
Z-projection image by “sum slices” command of ImageJ. In each acquired channel,
210
single cell area and mean fluorescence intensities (MFI, arbitrary unit) were measured.
211
The MFI of Alexa Fluor 488 and 568 channels were evaluated as total expression levels
212
of HA and NA in each cell, respectively. The total fluorescence intensity (MFI × area)
213
of HA divided by that of NA was evaluated as the expression ratio of HA-to-NA. The
214
cells (39-45 cells) were subjected to analysis of HA distribution patterns (either apical
215
PM alone or PMs plus cytoplasmic compartments). Sparse MDCK cells (1×105
216
cells/well) were also used in Fig. 3C.
217 218
TX-100 solubilization analysis and coimmunoprecipitation
219
293T cells were seeded into 12-well plates (5×105 cells/well) or
220
10-cm-diameter dishes (5×106 cells/dish). The cells were singly transfected with HA
221
and NA expression plasmids or cotransfected with a combination of HA and NA, HA
222
and M1, or HA and GFP-CD59 expression plasmids, respectively. At 24 hpt, the cells
223
were resuspended with cold TNE buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, and
224
150 mM NaCl) containing 1 mM DTT and protease inhibitor complete mini cocktail
225
(Roche). After brief sonication, the samples were treated with 1% TX-100 at 4°C or
226
37°C for 30 min and were centrifuged at 17,400×g for 30 min at 4°C to separate the
227
soluble and insoluble fractions. For TX-100 solubilization analysis, both the
228
supernatants and pellets were adjusted to be the same volume with TNE buffer and
229
analyzed by Western blotting. For coimmunoprecipitation, the supernatants were mixed
230
with 10 μg of anti-HA mAb12-1G6 and were incubated at 4°C for 90 min. The
231
supernatants were subsequently mixed with pre-blocked Protein G-Sepharose-beads
232
(GE Healthcare) and were incubated at 4°C for 60 min. After washing with TNE
233
containing 0.1% TX-100 three times, immunoprecipitates were eluted from the beads by
234
boiling with SDS sample buffer and were analyzed by Western blotting with sheep
235
anti-NA Ab (R&D Systems), rabbit anti-M1 polyclonal Ab (25). GFP-CD59 was
236
detected with mouse anti-GFP Ab (Sigma).
237
238
In situ proximity ligation assay (PLA)
239
In situ PLA was performed according to the manufacturer’s instructions (Olink
240
Biosciences). Briefly, polarized MDCK cells were either transfected with combinations
241
of protein expression plasmids or were infected with virus. At 9 hpt or 3.5 or 5 h
242
post-infection (hpi), the cells were fixed with 4% PFA, and permeabilized with 0.5%
243
TX-100. After blocking, the cells were incubated with mouse anti-HA mAb12-1G6,
244
rabbit anti-NA Ab, mouse anti-GFP Ab or rabbit caveolin-1 Ab (Santa Cruz) for 1 h at
245
room temperature. After being washed, the cells were incubated with two
246
species-specific secondary antibodies conjugated with unique oligonucleotides (rabbit
247
PLUS and mouse MINUS) as PLA probes. Phospholyrated connector oligonucleotides
248
were hybridized to the PLA probes. If the PLA probes are present in close proximity
249
(
1
Title
2
Influenza A virus hemagglutinin and neuraminidase mutually accelerate their
3
apical targeting through clustering of lipid rafts.
4 5
Running title
6
Apical targeting of HA and NA via lipid raft
7 8
Takashi Ohkura,a Fumitaka Momose,a Reiko Ichikawa,a Kaoru Takeuchi,b and Yuko
9
Morikawaa
10
a
11
University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
12
b
13
of Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan
Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato Department of Environmental Microbiology, Division of Biomedical Science, Faculty
14 15
Corresponding author:
16
Yuko Morikawa
17
Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato
18
University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan.
19
Phone:
20
[email protected]
+81-3-5791-6129;
Fax:
+81-3-5791-6268;
E-mail:
21
Abstract
22 23
In polarized epithelial cells, influenza A virus hemagglutinin (HA) and
24
neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical
25
plasma membrane for viral assembly and budding. Previous studies have indicated that
26
the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA were
27
required for association with lipid rafts, but the raft dependencies of their apical
28
targeting are controversial. Here, we show that coexpression of HA with NA accelerated
29
their apical targeting through accumulation in lipid rafts. HA was targeted to the apical
30
even when expressed alone but the kinetics was much slower than that of HA in infected
31
cells. Coexpression experiments revealed that apical targeting of HA and NA was
32
accelerated by their coexpression. The apical targeting of HA was also accelerated by
33
coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and
34
the deletion of the CT in HA and NA that reduced their association with lipid rafts
35
abolished the acceleration of their apical transport, indicating that the lipid raft
36
association is essential for efficient apical trafficking of HA and NA. An in situ
37
proximity ligation assay (PLA) revealed that HA and NA were accumulated and
38
clustered in the cytoplasmic compartments, only when both were associated with lipid
39
rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings.
40
We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism
41
of the accelerated apical transport of HA and NA via clustering of lipid rafts.
42
43
Importance
44 45
Lipid rafts serve as sites for viral entry, particle assembly, and budding, leading
46
to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma
47
membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase
48
(NA) of influenza virus, key players for particle assembly, contain determinants for
49
apical sorting and lipid raft association. However, it remains to be elucidated how lipid
50
rafts contribute to the apical trafficking and budding. We investigated the relation of
51
lipid raft association of HA and NA to the efficiency of apical trafficking. We show that
52
coexpression of HA and NA induces their accumulation in lipid rafts and accelerates
53
their apical targeting and suggest that the accelerated apical transport likely occurs by
54
clustering of lipid rafts at the TGN. This finding provides the first evidence that two
55
different raft-associated viral proteins induce lipid raft clustering, thereby accelerating
56
apical trafficking of the viral proteins.
57
Introduction
58 59
Influenza virus is an enveloped, negative-stranded, segmented RNA virus
60
belonging to the Orthomixoviridae family. The virion consists of three integral
61
membrane proteins, hemagglutinin (HA), neuraminidase (NA), and ion channel protein
62
M2. A layer of matrix protein M1 is present underneath the lipid envelope and encases
63
viral ribonucleoprotein (vRNP) complexes. The influenza virus buds from the apical
64
plasma membrane (PM), which is divided by tight junctions in polarized epithelial cells
65
(1). It is considered that all viral components are targeted to the apical PM, where
66
particle budding occurs. HA, NA, and M2 are synthesized at the endoplasmic reticulum
67
(ER) and are transported to the apical PM through the trans-Golgi network (TGN). The
68
apical sorting signals were identified in the transmembrane domain (TMD) of both HA
69
and NA (2, 3). Many studies indicate that during the apical trafficking, HA and NA are
70
associated with lipid raft microdomains, which are enriched in cholesterol and
71
sphingolipids (3, 4), whereas M2 is excluded from these domains (5, 6). Several studies
72
also indicate that the TMD and the cytoplasmic tail (CT) of HA and NA are important
73
for their association with lipid rafts (3, 5, 7). It has been shown that, in the case of HA,
74
palmitoylation at three conserved cysteines in the TMD-CT region is required for
75
association with lipid rafts (8). A very recent study suggested that M2 was a key player
76
in influenza virus particle budding, which is endosomal protein sorting complex
77
required for transport (ESCRT)-independent (9).
78
Lipid rafts are thought to function as platforms for selective concentration of
79
raft-associated proteins to promote protein-protein interactions for their functions (10).
80
Lipid rafts have also been shown to play pivotal roles in apical trafficking in polarized
81
cells (11) and in signal transduction pathways, such as Ras signaling (12) and PIP2
82
signaling (13). It has been suggested that for influenza virus HA and NA, the
83
association with lipid rafts constitutes a part of the machinery necessary for apical
84
trafficking in polarized cells (14, 15). Previous studies have indicated that disruption of
85
lipid rafts by treatment with methyl-β-cyclodextrin (MβCD) and lovastatin delays the
86
TGN-to-apical PM trafficking of HA and missorts to the basolateral membrane, whereas
87
vesicular stomatitis virus G protein, a nonraft-associated basolateral marker, remained
88
unaffected (16), suggesting that raft association is required for apical transport of
89
proteins. However, a number of studies have indicated that some mutations in the HA
90
TMD and CT did not impair apical targeting of HA irrespective of whether or not they
91
caused significant reductions in the raft association (5, 7), suggesting that raft
92
association is not essential for apical transport of HA.
93
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are not
94
integral membrane proteins but are associated with lipid rafts through the GPI moieties.
95
In this group of proteins, the raft association is not a determinant for apical sorting
96
because both apical and basolateral GPI-APs are associated with lipid rafts (17).
97
Interestingly, only apical, but not basolateral, GPI-APs form oligomer complexes when
98
they are associated with lipid rafts. When oligomerization of GPI-APs was impaired by
99
mutations, the GPI-APs were missorted to the basolateral PM domain (17). A recent
100
model has suggested that the oligomerization of GPI-APs promotes their stabilization in
101
lipid rafts, leading to their incorporation into apical transport vesicles (10).
102
Influenza virus budding and release require the assembly of viral components,
103
which occurs either during their trafficking to the apical PM or at the stage of particle
104
budding (6, 18, 19). During the trafficking, all viral components, HA, NA, M2, M1, and
105
vRNP, either individually or in their complex forms, are targeted to the apical PM and
106
form a higher order of complex (7, 19, 20). Although the apical sorting determinants for
107
individual viral components have been relatively well studied (2, 3), the molecular
108
mechanisms and kinetics involved in their apical targeting have not been elucidated.
109
In this study, we focused on the kinetics of apical targeting of influenza virus
110
envelope proteins in polarized MDCK cells. We found that HA and NA or M1, but not
111
M2, mutually accelerated their apical PM targeting. Using the TMD-CT mutants of HA
112
and NA, we show that the association of HA and NA with lipid rafts is necessary for the
113
acceleration of their apical PM targeting. Our data indicate that HA and NA come into
114
close proximity (clustering) in lipid rafts upon coexpression. Our data also show that
115
clustering of lipid rafts upon coexpression of HA and NA, suggesting a possible
116
mechanism of accelerated apical transport of HA and NA via the clustering of lipid rafts.
117
Materials and methods
118 119
Viruses and plasmids The H141Y and E142Q mutations were introduced into the HA gene of the
120 121
influenza A/Puerto Rico/8/34 (PR8) virus by inverted PCR. This PR8 derivative was
122
referred to as the wild type (wt) strain in the present study. The authentic PR8 strain was
123
used as the parental virus. The wt and mutant PR8 viruses were generated by reverse
124
genetics system with PolI plasmids (pHH21) and protein expression plasmids, as
125
described previously (21). Briefly, 293T cells were transfected with PolI plasmids for
126
synthesis of each viral RNA segment and protein expression plasmids for PB1, PB2, PA,
127
NP, HA and NA. At 6 h post-transfection (hpt), the cell medium was replaced with
128
Opti-MEM I (Gibco) supplemented with 5 μg/ml acetyl trypsin and 0.3% bovine serum
129
albumin (BSA), and the cells were incubated for 2 or 3 days. Recovered viruses were
130
grown in Madin-Darby canine kidney (MDCK) cells stably expressing HA
131
(MDCK-HA) (the kind gift of Dr. Takizawa from the Institute of Microbial Chemistry,
132
Japan).
133
The HA and NA constructs with deletion of the CT (ΔCT-HA and ΔCT-NA) for
134
recovery of recombinant viruses have essentially been described elsewhere (22, 23). For
135
ΔCT-HA, three consecutive stop codons were placed at the end of the TMD-coding
136
sequence and the downstream nucleotide sequence was left intact. For ΔCT-NA, the
137
authentic start codon was mutated and a new start codon was created at the beginning of
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the TMD-coding sequence. The constructs were cloned into pHH21. The HA and NA
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constructs with alanine substitutions in the CT (HA557-559, HA560-563, HA564-566,
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NA2-3, NA4-6, and NA2-6) and TMD (HA533-535, HA550-552, NA7-10, and
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NA31-35), and those with cysteine-to-serine substitutions at three palmitoylation sites
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(HA-SSS), were generated by overlapping PCR and were cloned into pHH21. The
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viruses with similar alanine substitutions have been described previously (3, 5, 8).
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The open reading frames of the wt-HA, NA, M1, and M2 genes were cloned
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into the eukaryotic expression plasmid pCAGGS, respectively. The open reading frames
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of the HA and NA constructs with deletion of the CT and those with the alanine
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substitutions were similarly cloned into pCAGGS, respectively. The cDNAs encoding
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CD59, the 75 kDa neurotrophin receptor (p75), and their GFP-tagged versions
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(GFP-CD59 and p75-GFP) were also cloned into pCAGGS.
150 151
Cell culture, infection, and DNA transfection
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MDCK and 293T cells were maintained in Dulbecco’s modified Eagle’s
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medium (Sigma) supplemented with 10% fetal bovine serum. MDCK cells were seeded
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into 12-well plates (1×106 cells/well) and were polarized by 12 h-incubation. The
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polarized MDCK cells were used throughout this study. The MDCK cells were infected
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with the wt virus at a multiplicity of infection (MOI) of 0.1 or 1 for 1 h. Transfection of
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DNA was carried out using Lipofectamine LTX (Invitrogen). To synchronize protein
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expression from plasmids, DNA-Lipofectamine complexes were sedimented by plate
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centrifugation at 250 × g for 5 min. In some experiments, MDCK cells were transfected
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with DNA and subsequently superinfected with the authentic PR8 virus.
161 162
Virus growth and plaque assay
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MDCK cells were inoculated with virus at an MOI of 0.1 in Opti-MEM I
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supplemented with 0.3% BSA for 1 h at 37°C. After being washed, the cells were
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incubated in Opti-MEM I supplemented with 5 μg/ml acetyl trypsin and 0.3% BSA. The
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culture medium was harvested at various time points and was subjected to plaque assay
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on MDCK or MDCK-HA cells.
168 169
Cholesterol depletion
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For the inhibition of cholesterol synthesis, 293T and MDCK cells were
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pre-treated with 8 μM lovastatin (Merck) for 12 h. After transfection, the cells were
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incubated in the presence of 8 μM lovastatin for 12 or 24 h. The cells were further
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treated with 5 or 10 mM methyl-β-cyclodextrin (MβCD) (Sigma) in the presence of 8
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μM lovastatin for 1 h.
175 176
Indirect immunofluorescence assay
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Polarized MDCK cells were grown on coverslips in 12-well plates and were
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either infected with virus or transfected with protein expression plasmids. The cells
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were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton
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X-100 (TX-100). Following blocking, the cells were incubated with primary antibodies
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(Abs) and subsequently with secondary Abs conjugated with Alexa Fluor 488, 568, or
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647 (Molecular Probes). The following Abs were used as primary Abs: mouse anti-HA
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mAb12-1G6 (24), mouse anti-HA Ab (Takara), rabbit anti-NA Ab (Sino Biological),
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sheep anti-NA Ab (R&D Systems), rabbit anti-M1 polyclonal Ab (25), mouse anti-M2
185
Ab (Santa Cruz), mouse anti-NP mAb61A5 (26), mouse anti-CD59 Ab (Abcam), rabbit
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anti-TGN46 Ab (Abcam), rabbit anti-ZO-3 Ab (Invitrogen), and rabbit anti-caveolin-1
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Ab (Santa Cruz). For costaining with HA and M2 or vRNP, anti-HA mAb12-1G6 was
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pre-labeled by using the Zenon Alexa Fluor 488 mouse IgG1 labeling kit (Invitrogen),
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and the mouse anti-HA (Takara), anti-M2, and anti-NP Abs were similarly pre-labeled
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with Alexa Fluor 568, as described previously (24). Nuclear staining was carried out
191
with TO-PRO-3 (Molecular Probes) or DAPI (Molecular Probes). The cells were
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observed with a laser scanning confocal microscope (TCS-SP5II AOBS; Leica).
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Confocal images were collected at 0.5-μm intervals along the Z-axis. Reconstitution of
194
an X-Z plane was processed using ImageJ software (27). Fifty antigen-positive cells
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were observed in each experiment and patterns of antigen distribution were analyzed.
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For quantitative analysis of HA and NA expression, polarized MDCK cells on
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coverslips in 12-well plates (1×106 cells/well) were singly transfected with an HA
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expression plasmid or cotransfected with the HA and an NA expression plasmids. After
199
fixation with 4% PFA, the cells were incubated with mouse anti-HA Ab (Takara) (for
200
HA on the apical PM). The cells were re-fixed with 4% PFA and were permeabilized
201
with 0.5% TX-100. The cells were incubated with rabbit anti-NA Ab (Sino Biological)
202
(for total NA) and subsequently with anti-mouse IgG conjugated with Alexa Fluor 647
203
and anti-rabbit IgG conjugated with Alexa Fluor 568. For total HA, the cells were
204
re-fixed with 4% PFA and were blocked with nonspecific mouse Ig. The cells were
205
finally incubated with anti-HA mAb12-1G6 pre-labeled by using the Zenon Alexa Fluor
206
488 mouse IgG labeling kit (Invitrogen). Nuclear staining was carried out with DAPI
207
(Molecular Probes). Confocal images were collected at 0.5-μm intervals along the
208
Z-axis. In each cell, the sum of intensity values of a Z-stack was calculated as a
209
Z-projection image by “sum slices” command of ImageJ. In each acquired channel,
210
single cell area and mean fluorescence intensities (MFI, arbitrary unit) were measured.
211
The MFI of Alexa Fluor 488 and 568 channels were evaluated as total expression levels
212
of HA and NA in each cell, respectively. The total fluorescence intensity (MFI × area)
213
of HA divided by that of NA was evaluated as the expression ratio of HA-to-NA. The
214
cells (39-45 cells) were subjected to analysis of HA distribution patterns (either apical
215
PM alone or PMs plus cytoplasmic compartments). Sparse MDCK cells (1×105
216
cells/well) were also used in Fig. 3C.
217 218
TX-100 solubilization analysis and coimmunoprecipitation
219
293T cells were seeded into 12-well plates (5×105 cells/well) or
220
10-cm-diameter dishes (5×106 cells/dish). The cells were singly transfected with HA
221
and NA expression plasmids or cotransfected with a combination of HA and NA, HA
222
and M1, or HA and GFP-CD59 expression plasmids, respectively. At 24 hpt, the cells
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were resuspended with cold TNE buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, and
224
150 mM NaCl) containing 1 mM DTT and protease inhibitor complete mini cocktail
225
(Roche). After brief sonication, the samples were treated with 1% TX-100 at 4°C or
226
37°C for 30 min and were centrifuged at 17,400×g for 30 min at 4°C to separate the
227
soluble and insoluble fractions. For TX-100 solubilization analysis, both the
228
supernatants and pellets were adjusted to be the same volume with TNE buffer and
229
analyzed by Western blotting. For coimmunoprecipitation, the supernatants were mixed
230
with 10 μg of anti-HA mAb12-1G6 and were incubated at 4°C for 90 min. The
231
supernatants were subsequently mixed with pre-blocked Protein G-Sepharose-beads
232
(GE Healthcare) and were incubated at 4°C for 60 min. After washing with TNE
233
containing 0.1% TX-100 three times, immunoprecipitates were eluted from the beads by
234
boiling with SDS sample buffer and were analyzed by Western blotting with sheep
235
anti-NA Ab (R&D Systems), rabbit anti-M1 polyclonal Ab (25). GFP-CD59 was
236
detected with mouse anti-GFP Ab (Sigma).
237
238
In situ proximity ligation assay (PLA)
239
In situ PLA was performed according to the manufacturer’s instructions (Olink
240
Biosciences). Briefly, polarized MDCK cells were either transfected with combinations
241
of protein expression plasmids or were infected with virus. At 9 hpt or 3.5 or 5 h
242
post-infection (hpi), the cells were fixed with 4% PFA, and permeabilized with 0.5%
243
TX-100. After blocking, the cells were incubated with mouse anti-HA mAb12-1G6,
244
rabbit anti-NA Ab, mouse anti-GFP Ab or rabbit caveolin-1 Ab (Santa Cruz) for 1 h at
245
room temperature. After being washed, the cells were incubated with two
246
species-specific secondary antibodies conjugated with unique oligonucleotides (rabbit
247
PLUS and mouse MINUS) as PLA probes. Phospholyrated connector oligonucleotides
248
were hybridized to the PLA probes. If the PLA probes are present in close proximity
249
(
