JVI Accepts, published online ahead of print on 25 June 2014 J. Virol. doi:10.1128/JVI.01117-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine
2
Reproductive and Respiratory Syndrome Virus Entry
3 4
Longjun Guo, Junwei Niu, Haidong Yu, Weihong Gu, Ren Li, Xiaolei Luo, Mingming
5
Huang, Zhijun Tian, Li Feng, Yue Wang#
6 7
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,
8
Chinese Academy of Agricultural Sciences, Harbin, China
9 10
Running Title: Metalloprotease ADAM17 regulates PRRSV Entry
11 12
#
13
Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural
14
Sciences, Harbin 150001; TEL: +86 18946061091; FAX: +86 451-51997166; Email:
15
[email protected]; [email protected]
Address correspondence to Yue Wang, State Key Laboratory of Veterinary
16 17 18
Abstract word count: 181
19
Text word count: 4,443
20
1
21
ABSTRACT
22
As a consequence of their effects on ectodomain shedding, members of the A disintegrin
23
and metalloprotease (ADAM) family have been implicated in the control of various
24
cellular processes. Although ADAM family members are also involved in cancer,
25
inflammation, and other pathologies, it’s unclear whether they affect porcine reproductive
26
and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first
27
time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine
28
alveolar macrophages (PAMs). We also demonstrate that inhibition of ADAM17 up-
29
regulates membrane CD163 expression, a putative PRRSV receptor that is exogenously
30
expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore,
31
overexpression of ADAM17 induced down-regulation of CD163 expression and a
32
reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific
33
siRNA resulted in up-regulation of CD163 expression with a corresponding increase in
34
PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV non-
35
permissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate
36
that ADAM17 down-regulates CD163 expression and hinders PRRSV entry. Hence,
37
down-regulation of ADAM17 particular substrates may be an additional component of
38
the anti-infection defenses.
39 40
IMPORTANCE
41
ADAM17 is one of the important membrane-associated metalloproteases that mediate
42
various cellular events as well as inflammation, cancer, and other pathologies. Here, we
43
investigate for the first time the role of the metalloprotease ADAM17 in PRRSV
44
infection. By using inhibitor and genetic modification methods, we demonstrate that 2
45
ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More
46
specifically, ADAM 17 mediates the down-regulation of the PRRSV cellular receptor
47
CD163. The reduction in CD163 expression represents another component of the anti-
48
infection response initiated by ADAM17.
49
3
50
INTRODUCTION
51
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped,
52
positive-stranded RNA virus in the family Arteriviridae and the order Nidovirales (1).
53
Based on genetic differences, PRRSV has been divided into European strains (EU,
54
represented by Lelystad) and North American strains (NA, represented by VR2332) (2,
55
3). Porcine reproductive and respiratory syndrome (PRRS) is characterized by an acute
56
viral infection that leads to respiratory problems in growing pigs and reproductive failure
57
in sows. In China, a highly pathogenic strain of PRRSV (HP-PRRSV) was isolated and
58
identified in 2006. HP-PRRSV, which has a 30-amino-acid depletion in nonstructural
59
protein 2 (nsp2), has caused great economic losses for the swine industry (4, 5).
60
PRRSV has a tropism for cells of the monocytic lineage such as porcine alveolar
61
macrophages (PAMs). To date, researchers have identified two essential viral receptors
62
that mediate PRRSV entry and uncoating; these receptors are sialoadhesin (CD169 or
63
Siglec-1) and the scavenger receptor CD163 (6-8). In primary macrophages, these two
64
receptors are expressed at high level, which explains why PAMs are highly susceptible to
65
PRRSV infection. In addition, MA-104 (African green monkey kidney cell) and its
66
derivate Marc-145 are well-characterized cell lines that have been used to sustain PRRSV
67
in culture. CD163 has been identified as a key molecule in PRRSV entry into Marc-145
68
cells even though these cells do not belong to the monocyte-macrophage lineage (7).
69
Several papers have reported that the expression of CD163 alone in non-permissive cells
70
leads to high titers of progeny virus (7, 9-13), which indicates that CD163 plays a major
71
role in PRRSV entry.
4
72
As noted earlier, CD163 belongs to the scavenger receptor cysteine-rich superfamily
73
(SRCR) (14). The expression of CD163 is tightly regulated by a variety of factors and is
74
down-regulated by proinflammatory cytokine TNFα and the cross-linking of Fcγ receptor
75
(15, 16). Previous reports revealed that a disintegrin and metalloprotease 17 (ADAM17)
76
mediates human CD163 down-regulation and production of the soluble form of CD163
77
(17-19). As a member of the metalloprotease family, ADAM17 is the best-studied
78
ADAM sheddase (20); ADAM sheddases can cleave various cell surface proteins,
79
typically at a juxtamembrane site, resulting in the down-regulation of membrane proteins
80
expression and the release a soluble form of ectodomain fragment (21, 22). The list of
81
cell surface proteins known to be cleaved by ADAM17 is still growing, and most of its
82
substrates, including TNFα, TNF-RI, TNF-RII, and L-selectin (23, 24), play important
83
roles in modulating inflammation, indicating that ADAM17 has a critical role in host
84
immune response. In spite of the central role ADAM17 in many biological processes,
85
how this metalloprotease governs PRRSV infection remains unstudied.
86
Here, we examined the role of ADAM17 on PRRSV infection. We demonstrate that
87
ADAM17 directly reduces PRRSV entry as a consequence of its proteolytic activity.
88
ADAM17 down-regulated the exogenous and endogenous expression of CD163, a
89
PRRSV putative receptor, in different cells. Ablation of ADAM17 expression using a
90
specific inhibitor or small interfering RNA duplexes reduced CD163 down-regulation
91
and enhanced PRRSV infection, whereas overexpression of ADAM17 increased CD163
92
down-regulation and suppressed PRRSV infection. Taken together, these data provide
93
direct evidence that the metalloprotease ADAM17 regulates PRRSV entry by modifying
94
the expression of membrane CD163.
5
95 96
MATERIALS AND METHODS
97 98
Cells and viruses
99
The following cells were maintained in Dulbecco’s minimum essential medium
100
(DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS,
101
Thermofisher): Marc-145, a monkey kidney cell line subcloned from MA-104; BHK-21,
102
a cell line derived from baby Syrian hamster kidney; and 293T. Primary porcine alveolar
103
macrophages (PAMs) were freshly harvested from 6-week-old specific-pathogen-free
104
(SPF) pigs and were maintained in DMEM with 10% heat-inactivated FBS and
105
penicillin-streptomycin. The animal experiment was approved by Harbin Veterinary
106
Research Institute and performed in accordance with animal ethics guidelines and
107
approved protocols. The animal ethics committee approval number is Heilongjiang-
108
SYXK-2006-032. All cells were cultured in a humidified atmosphere at 37°C and 5%
109
CO2. The type II PRRSV strain HuN4 (GenBank accession number EF635006), which is
110
a highly pathogenic PRRSV strain that was isolated in China, was grown and titrated in
111
Marc-145 cells as described before (25) and was stored at -80°C.
112 113
Immunofluorescence assay (IFA) and viral plaque assay
114
After various treatments, the cell monolayer was washed with phosphate-buffered
115
saline (PBS) and inoculated with PRRSV HuN4 at a multiplicity of infection (MOI) of
116
0.1 for 60 min at 37°C with rocking. After removal of the inoculum, the cell monolayer
117
was washed twice with PBS and covered with the medium. At 24 h post-inoculation, cells
6
118
were washed twice with PBS, fixed in 33.3% acetone for 30 min at room temperature,
119
and dried. Fixed cells were incubated with fluorescein isothiocyanate (FITC)-conjugated
120
anti-PRRSV N protein monoclonal antibody (mAb) SDOW17 (Rural Technologies,
121
USA) for 30 min at 37°C. After the cells were washed three times with PBS at 5 min
122
intervals, the fluorescence was visualized with an Olympus inverted fluorescence
123
microscope equipped with a camera, and the number of susceptible cells was counted. A
124
plaque assay was performed as previously described with slight modifications (26).
125
Typically, Marc-145 cells cultured in a 6-well-plate until 90% confluency were
126
inoculated with PRRSV HuN4 at a MOI of 0.1. After 60 min of incubation, cell
127
monolayers were washed and then overlaid with an equivalent volume of 2×DMEM
128
containing 10% FBS and 3.0% low-melting-temperature agarose (Cambrex, Rockland,
129
ME). When the agarose overlay solidified, the plates were inverted and moved to a
130
humidified incubator at 37 °C and 5% CO2. When plaques had fully developed, plates
131
were stained with crystal violet (5% [wt/vol] in 20% ethanol).
132 133
Transfection of ADAM17 and CD163
134
The genes of ADAM17 and CD163 were amplified from mouse and porcine
135
macrophages cDNA, respectively, and cloned into the pCAGGS vector (Addgene, USA).
136
The Flag tag (DYKDDDDK) was fused at the carboxyl terminus of ADAM17 by PCR
137
using the primers listed in Table 1. The nucleotide sequences of the plasmids encoding
138
ADAM17 and CD163 were determined to ensure that the correct clones were used in the
139
study. Marc-145 or BHK-21 cells were transfected with 2 µg of target plasmid
140
pCAGGS/ADAM17, pCAGGS/CD163, and vector control pCAGGS using X-
7
141
tremeGENE transfection reagent (Roche). At 24 h post-transfection, cells were inoculated
142
with PRRSV HuN4 at a MOI of 0.1, and the infection of PRRSV was detected by IFA as
143
described above. Also at 24 h post-transfection, cells were lysed in RIPA buffer
144
(Beyotime, Nantong, China) for western-blot analysis of the expression of ADAM17 or
145
CD163.
146 147
Metalloprotease ADAM17 inhibition assay
148
To investigate the role of ADAM17 in altering PRRSV entry by regulating viral
149
cellular receptors, such as CD163 and CD169, we used batimastat (BB94, Sigma-
150
Aldrich, St. Louis, MO) (27) as a metalloprotease ADAM17 inhibitor. The cytotoxicity
151
effect of BB94 was determined by trypan blue staining with three replicates for each
152
inhibitor concentration (2, 5, 10, 20, and 50 µM). Concentrations < 10 µM were non-
153
toxic (see Results), and cells were therefore incubated with 2 µM BB94 for 24 h before
154
they were inoculated with PRRSV HuN4 at a MOI of 0.1. PRRSV infection was
155
examined by IFA and plaque assay as described above. In addition, the surface
156
expression level of CD163 or CD169 on some cells was assessed using specific
157
antibodies by flow cytometry. For BHK-21 cells, cells were pre-incubated with BB94 for
158
24 h and were then transfected with pCAGGS/CD163. At 24 h post-transfection, CD163
159
expression on the cell surface was detected by flow cytometry. In addition, at 24 h post-
160
transfection of pCAGGS/CD163, BHK-21 cells were inoculated with PRRSV at a MOI
161
of 0.1, and PRRSV infection was assessed by IFA. To rule out the effect of intracellular
162
inhibition of BB94 and to ensure that the metalloprotease ADAM17 alters PRRSV entry
163
rather than PRRSV replication, a Marc-145 monolayer was inoculated with PRRSV at a
8
164
MOI of 0.1 for 60 min and was then incubated in a medium containing the ADAM17
165
inhibitor BB94. At 24 h post-inoculation, the infection of PRRSV was detected by IFA.
166
For all experiments, cells were treated with the appropriate amount of DMSO (carrier) for
167
mock treatments.
168
To confirm ADAM17 function, siRNA duplexes were introduced to knock down
169
ADAM17 expression. Marc-145 cells were grown to ~60% confluency in an antibiotic-
170
free growth medium and transfected with siRNA duplexes (ON-TARGET-plus
171
SMARTpoolsiRNA oligonucleotides, Sigma-Aldrich) using Dharmafect-2 transfection
172
reagent (Thermo Scientific) according to the manufacturer’s instructions. Cells were
173
separately transfected with each of three siRNA duplexes targeted to ADAM17 and with
174
control siRNA duplexes targeted to a scramble sequence (Table 2) at the concentration of
175
25 nM and 100 nM. Twenty-four hours after transfection, cell lysates were prepared and
176
assayed for specific gene silencing by real-time PCR and western-blot. In some
177
experiments, PRRSV infection at a MOI of 0.1 was performed 24 h post-transfection.
178 179
SDS-PAGE and western-blot
180
Cell were detergent-lysed as previously described (28). Typically, samples were
181
separated by SDS-PAGE under reducing conditions and transferred onto a PVDF
182
membrane. Membranes were blocked with 2% BSA in PBS for 60 min and then
183
incubated with a primary antibody for 60 min. After they were washed three times with
184
PBS + 0.05% Tween-20, the membranes were incubated with IRDye-conjugated
185
secondary antibody (Li-Cor Biosciences, Lincoln, NE) diluted in washing buffer for 60
186
min. Membranes were washed as described above and were scanned and analyzed with
9
187
an Odyssey instrument (Odyssey infrared imaging system; Li-Cor Biosciences) according
188
to the manufacturer’s instructions. For detection of ADAM17 expression, the PVDF
189
membrane was probed with rabbit polyclonal antibody to ADAM17 (Abcam, MA, USA).
190
The blotting antibody for CD163 was raised in rabbit and stocked in our laboratory. The
191
mAb M2 (Sigma-Aldrich, St. Louis, MO) was used to detect the expression of Flag-
192
tagged proteins. Anti-β-actin mAb (C4) was purchased from Santa Cruz Biotechnology
193
(Santa Cruz, CA).
194 195
Flow cytometry
196
Flow cytometric analyses were performed on a FACSAria instrument (BD
197
Biosciences) as described previously (29). Allophycocyanin (APC) conjugated mouse
198
anti-human CD163 mAb (BioLegend) detects CD163 expressed on Marc-145 cells.
199
Mouse anti-pig CD163 and anti-pig CD169 mAbs were purchased from AbD Serotec.
200
Mouse anti-PRRSV N protein mAb SDOW17 labeled with FITC (SDOW17-F) was
201
purchased from Rural Technologies. For intracellular PRRSV detection, cells were
202
pretreated with a fixation/permeabilization kit following the manufacturer’s protocol
203
(eBioscience) and were then stained with SDOW17-F. Isotype-matched negative control
204
mAbs were used to evaluate levels of nonspecific staining. Typically, 10,000 labeled
205
cells were analyzed. All samples were analyzed with FlowJo 8.7 (Tree Star) and FACS
206
Diva (BD Biosciences).
207 208
Quantitative RT-PCR
10
209
At 24 h post-transfection of siRNA, total RNA was extracted from cells using the
210
RNeasy RNA purification kit (Qiagen) to generate cDNA with oligo (dT) primers using
211
Superscript® II reverse transcriptase (Invitrogen, USA). Quantitative RT-PCR reactions
212
were conducted in triplicate using SYBR premix Ex Taq (TaKaRa) and 10 μM specific
213
primers (Table 1). Relative quantification was performed by the cycle threshold (∆∆CT)
214
method (30). Briefly, CT values were normalized to glyceraldehyde 3-phosphate
215
dehydrogenase mRNA (GAPDH, internal standard), and ∆CT was determined by the
216
formula ∆CT = CT (ADM17) - CT (GAPDH). Fold-change was determined by 2-∆∆CT,
217
where ∆∆CT=∆CT (siRNA1-3#) -∆CT (siControl).
218 219
Statistical analysis Values are expressed as means SD. Data were analyzed with Student’s t-test in
220 221
Excel. A p value of 60% as indicated by
656
the relative levels of ADAM17 mRNA to GAPDH mRNA. (D) Detergent lysate from
657
ADAM17-specific siRNA transfected Marc-145 cells was subjected to reducing SDS-
658
PAGE and immunoblotting with antibodies to ADAM17 or β-actin (loading control). (E)
659
ADAM17-specific siRNA enhances PRRSV infection. Marc-145 cells were transfected
660
with 2# siRNA duplexes or control siRNA at 100 nM for 24 h before they were
661
inoculated with PRRSV HuN4. At 24 h post-inoculation, the cell monolayer was fixed,
662
and the number of PRRSV-positive cells was determined by FITC-conjugated mAb
663
SDOW17. In A, C, and E, representative values from three separate experiments are
664
shown, and each value represents the mean ± SD of three separate experiments.
665
0.05. The p value was calculated using Student's t-test.
＊,
p
1
Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine
2
Reproductive and Respiratory Syndrome Virus Entry
3 4
Longjun Guo, Junwei Niu, Haidong Yu, Weihong Gu, Ren Li, Xiaolei Luo, Mingming
5
Huang, Zhijun Tian, Li Feng, Yue Wang#
6 7
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,
8
Chinese Academy of Agricultural Sciences, Harbin, China
9 10
Running Title: Metalloprotease ADAM17 regulates PRRSV Entry
11 12
#
13
Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural
14
Sciences, Harbin 150001; TEL: +86 18946061091; FAX: +86 451-51997166; Email:
15
[email protected]; [email protected]
Address correspondence to Yue Wang, State Key Laboratory of Veterinary
16 17 18
Abstract word count: 181
19
Text word count: 4,443
20
1
21
ABSTRACT
22
As a consequence of their effects on ectodomain shedding, members of the A disintegrin
23
and metalloprotease (ADAM) family have been implicated in the control of various
24
cellular processes. Although ADAM family members are also involved in cancer,
25
inflammation, and other pathologies, it’s unclear whether they affect porcine reproductive
26
and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first
27
time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine
28
alveolar macrophages (PAMs). We also demonstrate that inhibition of ADAM17 up-
29
regulates membrane CD163 expression, a putative PRRSV receptor that is exogenously
30
expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore,
31
overexpression of ADAM17 induced down-regulation of CD163 expression and a
32
reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific
33
siRNA resulted in up-regulation of CD163 expression with a corresponding increase in
34
PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV non-
35
permissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate
36
that ADAM17 down-regulates CD163 expression and hinders PRRSV entry. Hence,
37
down-regulation of ADAM17 particular substrates may be an additional component of
38
the anti-infection defenses.
39 40
IMPORTANCE
41
ADAM17 is one of the important membrane-associated metalloproteases that mediate
42
various cellular events as well as inflammation, cancer, and other pathologies. Here, we
43
investigate for the first time the role of the metalloprotease ADAM17 in PRRSV
44
infection. By using inhibitor and genetic modification methods, we demonstrate that 2
45
ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More
46
specifically, ADAM 17 mediates the down-regulation of the PRRSV cellular receptor
47
CD163. The reduction in CD163 expression represents another component of the anti-
48
infection response initiated by ADAM17.
49
3
50
INTRODUCTION
51
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped,
52
positive-stranded RNA virus in the family Arteriviridae and the order Nidovirales (1).
53
Based on genetic differences, PRRSV has been divided into European strains (EU,
54
represented by Lelystad) and North American strains (NA, represented by VR2332) (2,
55
3). Porcine reproductive and respiratory syndrome (PRRS) is characterized by an acute
56
viral infection that leads to respiratory problems in growing pigs and reproductive failure
57
in sows. In China, a highly pathogenic strain of PRRSV (HP-PRRSV) was isolated and
58
identified in 2006. HP-PRRSV, which has a 30-amino-acid depletion in nonstructural
59
protein 2 (nsp2), has caused great economic losses for the swine industry (4, 5).
60
PRRSV has a tropism for cells of the monocytic lineage such as porcine alveolar
61
macrophages (PAMs). To date, researchers have identified two essential viral receptors
62
that mediate PRRSV entry and uncoating; these receptors are sialoadhesin (CD169 or
63
Siglec-1) and the scavenger receptor CD163 (6-8). In primary macrophages, these two
64
receptors are expressed at high level, which explains why PAMs are highly susceptible to
65
PRRSV infection. In addition, MA-104 (African green monkey kidney cell) and its
66
derivate Marc-145 are well-characterized cell lines that have been used to sustain PRRSV
67
in culture. CD163 has been identified as a key molecule in PRRSV entry into Marc-145
68
cells even though these cells do not belong to the monocyte-macrophage lineage (7).
69
Several papers have reported that the expression of CD163 alone in non-permissive cells
70
leads to high titers of progeny virus (7, 9-13), which indicates that CD163 plays a major
71
role in PRRSV entry.
4
72
As noted earlier, CD163 belongs to the scavenger receptor cysteine-rich superfamily
73
(SRCR) (14). The expression of CD163 is tightly regulated by a variety of factors and is
74
down-regulated by proinflammatory cytokine TNFα and the cross-linking of Fcγ receptor
75
(15, 16). Previous reports revealed that a disintegrin and metalloprotease 17 (ADAM17)
76
mediates human CD163 down-regulation and production of the soluble form of CD163
77
(17-19). As a member of the metalloprotease family, ADAM17 is the best-studied
78
ADAM sheddase (20); ADAM sheddases can cleave various cell surface proteins,
79
typically at a juxtamembrane site, resulting in the down-regulation of membrane proteins
80
expression and the release a soluble form of ectodomain fragment (21, 22). The list of
81
cell surface proteins known to be cleaved by ADAM17 is still growing, and most of its
82
substrates, including TNFα, TNF-RI, TNF-RII, and L-selectin (23, 24), play important
83
roles in modulating inflammation, indicating that ADAM17 has a critical role in host
84
immune response. In spite of the central role ADAM17 in many biological processes,
85
how this metalloprotease governs PRRSV infection remains unstudied.
86
Here, we examined the role of ADAM17 on PRRSV infection. We demonstrate that
87
ADAM17 directly reduces PRRSV entry as a consequence of its proteolytic activity.
88
ADAM17 down-regulated the exogenous and endogenous expression of CD163, a
89
PRRSV putative receptor, in different cells. Ablation of ADAM17 expression using a
90
specific inhibitor or small interfering RNA duplexes reduced CD163 down-regulation
91
and enhanced PRRSV infection, whereas overexpression of ADAM17 increased CD163
92
down-regulation and suppressed PRRSV infection. Taken together, these data provide
93
direct evidence that the metalloprotease ADAM17 regulates PRRSV entry by modifying
94
the expression of membrane CD163.
5
95 96
MATERIALS AND METHODS
97 98
Cells and viruses
99
The following cells were maintained in Dulbecco’s minimum essential medium
100
(DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS,
101
Thermofisher): Marc-145, a monkey kidney cell line subcloned from MA-104; BHK-21,
102
a cell line derived from baby Syrian hamster kidney; and 293T. Primary porcine alveolar
103
macrophages (PAMs) were freshly harvested from 6-week-old specific-pathogen-free
104
(SPF) pigs and were maintained in DMEM with 10% heat-inactivated FBS and
105
penicillin-streptomycin. The animal experiment was approved by Harbin Veterinary
106
Research Institute and performed in accordance with animal ethics guidelines and
107
approved protocols. The animal ethics committee approval number is Heilongjiang-
108
SYXK-2006-032. All cells were cultured in a humidified atmosphere at 37°C and 5%
109
CO2. The type II PRRSV strain HuN4 (GenBank accession number EF635006), which is
110
a highly pathogenic PRRSV strain that was isolated in China, was grown and titrated in
111
Marc-145 cells as described before (25) and was stored at -80°C.
112 113
Immunofluorescence assay (IFA) and viral plaque assay
114
After various treatments, the cell monolayer was washed with phosphate-buffered
115
saline (PBS) and inoculated with PRRSV HuN4 at a multiplicity of infection (MOI) of
116
0.1 for 60 min at 37°C with rocking. After removal of the inoculum, the cell monolayer
117
was washed twice with PBS and covered with the medium. At 24 h post-inoculation, cells
6
118
were washed twice with PBS, fixed in 33.3% acetone for 30 min at room temperature,
119
and dried. Fixed cells were incubated with fluorescein isothiocyanate (FITC)-conjugated
120
anti-PRRSV N protein monoclonal antibody (mAb) SDOW17 (Rural Technologies,
121
USA) for 30 min at 37°C. After the cells were washed three times with PBS at 5 min
122
intervals, the fluorescence was visualized with an Olympus inverted fluorescence
123
microscope equipped with a camera, and the number of susceptible cells was counted. A
124
plaque assay was performed as previously described with slight modifications (26).
125
Typically, Marc-145 cells cultured in a 6-well-plate until 90% confluency were
126
inoculated with PRRSV HuN4 at a MOI of 0.1. After 60 min of incubation, cell
127
monolayers were washed and then overlaid with an equivalent volume of 2×DMEM
128
containing 10% FBS and 3.0% low-melting-temperature agarose (Cambrex, Rockland,
129
ME). When the agarose overlay solidified, the plates were inverted and moved to a
130
humidified incubator at 37 °C and 5% CO2. When plaques had fully developed, plates
131
were stained with crystal violet (5% [wt/vol] in 20% ethanol).
132 133
Transfection of ADAM17 and CD163
134
The genes of ADAM17 and CD163 were amplified from mouse and porcine
135
macrophages cDNA, respectively, and cloned into the pCAGGS vector (Addgene, USA).
136
The Flag tag (DYKDDDDK) was fused at the carboxyl terminus of ADAM17 by PCR
137
using the primers listed in Table 1. The nucleotide sequences of the plasmids encoding
138
ADAM17 and CD163 were determined to ensure that the correct clones were used in the
139
study. Marc-145 or BHK-21 cells were transfected with 2 µg of target plasmid
140
pCAGGS/ADAM17, pCAGGS/CD163, and vector control pCAGGS using X-
7
141
tremeGENE transfection reagent (Roche). At 24 h post-transfection, cells were inoculated
142
with PRRSV HuN4 at a MOI of 0.1, and the infection of PRRSV was detected by IFA as
143
described above. Also at 24 h post-transfection, cells were lysed in RIPA buffer
144
(Beyotime, Nantong, China) for western-blot analysis of the expression of ADAM17 or
145
CD163.
146 147
Metalloprotease ADAM17 inhibition assay
148
To investigate the role of ADAM17 in altering PRRSV entry by regulating viral
149
cellular receptors, such as CD163 and CD169, we used batimastat (BB94, Sigma-
150
Aldrich, St. Louis, MO) (27) as a metalloprotease ADAM17 inhibitor. The cytotoxicity
151
effect of BB94 was determined by trypan blue staining with three replicates for each
152
inhibitor concentration (2, 5, 10, 20, and 50 µM). Concentrations < 10 µM were non-
153
toxic (see Results), and cells were therefore incubated with 2 µM BB94 for 24 h before
154
they were inoculated with PRRSV HuN4 at a MOI of 0.1. PRRSV infection was
155
examined by IFA and plaque assay as described above. In addition, the surface
156
expression level of CD163 or CD169 on some cells was assessed using specific
157
antibodies by flow cytometry. For BHK-21 cells, cells were pre-incubated with BB94 for
158
24 h and were then transfected with pCAGGS/CD163. At 24 h post-transfection, CD163
159
expression on the cell surface was detected by flow cytometry. In addition, at 24 h post-
160
transfection of pCAGGS/CD163, BHK-21 cells were inoculated with PRRSV at a MOI
161
of 0.1, and PRRSV infection was assessed by IFA. To rule out the effect of intracellular
162
inhibition of BB94 and to ensure that the metalloprotease ADAM17 alters PRRSV entry
163
rather than PRRSV replication, a Marc-145 monolayer was inoculated with PRRSV at a
8
164
MOI of 0.1 for 60 min and was then incubated in a medium containing the ADAM17
165
inhibitor BB94. At 24 h post-inoculation, the infection of PRRSV was detected by IFA.
166
For all experiments, cells were treated with the appropriate amount of DMSO (carrier) for
167
mock treatments.
168
To confirm ADAM17 function, siRNA duplexes were introduced to knock down
169
ADAM17 expression. Marc-145 cells were grown to ~60% confluency in an antibiotic-
170
free growth medium and transfected with siRNA duplexes (ON-TARGET-plus
171
SMARTpoolsiRNA oligonucleotides, Sigma-Aldrich) using Dharmafect-2 transfection
172
reagent (Thermo Scientific) according to the manufacturer’s instructions. Cells were
173
separately transfected with each of three siRNA duplexes targeted to ADAM17 and with
174
control siRNA duplexes targeted to a scramble sequence (Table 2) at the concentration of
175
25 nM and 100 nM. Twenty-four hours after transfection, cell lysates were prepared and
176
assayed for specific gene silencing by real-time PCR and western-blot. In some
177
experiments, PRRSV infection at a MOI of 0.1 was performed 24 h post-transfection.
178 179
SDS-PAGE and western-blot
180
Cell were detergent-lysed as previously described (28). Typically, samples were
181
separated by SDS-PAGE under reducing conditions and transferred onto a PVDF
182
membrane. Membranes were blocked with 2% BSA in PBS for 60 min and then
183
incubated with a primary antibody for 60 min. After they were washed three times with
184
PBS + 0.05% Tween-20, the membranes were incubated with IRDye-conjugated
185
secondary antibody (Li-Cor Biosciences, Lincoln, NE) diluted in washing buffer for 60
186
min. Membranes were washed as described above and were scanned and analyzed with
9
187
an Odyssey instrument (Odyssey infrared imaging system; Li-Cor Biosciences) according
188
to the manufacturer’s instructions. For detection of ADAM17 expression, the PVDF
189
membrane was probed with rabbit polyclonal antibody to ADAM17 (Abcam, MA, USA).
190
The blotting antibody for CD163 was raised in rabbit and stocked in our laboratory. The
191
mAb M2 (Sigma-Aldrich, St. Louis, MO) was used to detect the expression of Flag-
192
tagged proteins. Anti-β-actin mAb (C4) was purchased from Santa Cruz Biotechnology
193
(Santa Cruz, CA).
194 195
Flow cytometry
196
Flow cytometric analyses were performed on a FACSAria instrument (BD
197
Biosciences) as described previously (29). Allophycocyanin (APC) conjugated mouse
198
anti-human CD163 mAb (BioLegend) detects CD163 expressed on Marc-145 cells.
199
Mouse anti-pig CD163 and anti-pig CD169 mAbs were purchased from AbD Serotec.
200
Mouse anti-PRRSV N protein mAb SDOW17 labeled with FITC (SDOW17-F) was
201
purchased from Rural Technologies. For intracellular PRRSV detection, cells were
202
pretreated with a fixation/permeabilization kit following the manufacturer’s protocol
203
(eBioscience) and were then stained with SDOW17-F. Isotype-matched negative control
204
mAbs were used to evaluate levels of nonspecific staining. Typically, 10,000 labeled
205
cells were analyzed. All samples were analyzed with FlowJo 8.7 (Tree Star) and FACS
206
Diva (BD Biosciences).
207 208
Quantitative RT-PCR
10
209
At 24 h post-transfection of siRNA, total RNA was extracted from cells using the
210
RNeasy RNA purification kit (Qiagen) to generate cDNA with oligo (dT) primers using
211
Superscript® II reverse transcriptase (Invitrogen, USA). Quantitative RT-PCR reactions
212
were conducted in triplicate using SYBR premix Ex Taq (TaKaRa) and 10 μM specific
213
primers (Table 1). Relative quantification was performed by the cycle threshold (∆∆CT)
214
method (30). Briefly, CT values were normalized to glyceraldehyde 3-phosphate
215
dehydrogenase mRNA (GAPDH, internal standard), and ∆CT was determined by the
216
formula ∆CT = CT (ADM17) - CT (GAPDH). Fold-change was determined by 2-∆∆CT,
217
where ∆∆CT=∆CT (siRNA1-3#) -∆CT (siControl).
218 219
Statistical analysis Values are expressed as means SD. Data were analyzed with Student’s t-test in
220 221
Excel. A p value of 60% as indicated by
656
the relative levels of ADAM17 mRNA to GAPDH mRNA. (D) Detergent lysate from
657
ADAM17-specific siRNA transfected Marc-145 cells was subjected to reducing SDS-
658
PAGE and immunoblotting with antibodies to ADAM17 or β-actin (loading control). (E)
659
ADAM17-specific siRNA enhances PRRSV infection. Marc-145 cells were transfected
660
with 2# siRNA duplexes or control siRNA at 100 nM for 24 h before they were
661
inoculated with PRRSV HuN4. At 24 h post-inoculation, the cell monolayer was fixed,
662
and the number of PRRSV-positive cells was determined by FITC-conjugated mAb
663
SDOW17. In A, C, and E, representative values from three separate experiments are
664
shown, and each value represents the mean ± SD of three separate experiments.
665
0.05. The p value was calculated using Student's t-test.
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p
