2011/01/17 1
2
SARS coronavirus papain-like protease suppressed interferon-α-induced 3
responses through down-regulation of ERK1-mediated signaling pathways 4
5 6
Shih-Wein Li 1,2 Chien-Chen Lai 2,3¶ Jia-Fong Ping1 Fuu-Jen Tsai3 7
Lei Wan3 Ying-Ju Lin 3 Szu-Hao Kung 4 Cheng-Wen Lin 1,5,6 * 8
9
1 Department of Medical Laboratory Science and Biotechnology, China Medical 10
University, Taichung, Taiwan 11
2 Institute of Molecular Biology, National Chung Hsing University, Taichung, 12
Taiwan 13
3 Department of Medical Genetics and Medical Research, China Medical University 14
Hospital, Taichung, Taiwan 15
4 Department of Biotechnology and Laboratory Science in Medicine, National Yang 16
Ming University, Taipei, Taiwan 17
5 Clinical Virology Laboratory, Department of Laboratory Medicine, China Medical 18
University Hospital, Taichung, Taiwan 19
6 Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan 20
21 22
Running title: SARS CoV PLpro suppressed ERK1/STAT1 signaling 23
24 25 26 27
¶Co-first author 28
*Corresponding author: Cheng-Wen Lin, PhD, Professor. Department of Medical 29
Laboratory Science and Biotechnology, China Medical University, No. 91, 30
Hsueh-Shih Road, Taichung 404, Taiwan 31
Fax: 886-4-22057414 32
Email: [email protected] 33
34
Abstract 35
SARS coronavirus (SARS-CoV) papain-like protease (PLpro), a 36
deubiquitinating enzyme, reportedly blocks polyI:C-induced activation of IRF3 and 37
NF-κB, reducing interferon (IFN) induction. This study investigated Type I IFN 38
antagonist mechanism of PLpro in human promonocytes. PLpro antagonized 39
IFNα-induced responses such as ISRE- and AP-1-driven promoter activation, PKR, 40
2’-5’-OAS, IL-6 and IL-8 expression, and STAT1(Tyr701), STAT1(Ser727) and c-Jun 41
phosphorylation. Proteomics approach demonstrated down-regulation of ERK1 and 42
up-regulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory 43
mechanism of PLpro on IFNα-induced responses. IFNα treatment significantly 44
induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent 45
decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of 46
ERK1 showed a relationship between ERK1 and ubiquitin proteasome signaling 47
pathways associated with IFN antagonism by PLpro. Combination treatment of IFNα 48
and proteasome inhibitor MG132 showed a time-dependent restoration of ERK1 49
protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in 50
PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment 51
significantly reduced IFNα-induced ERK1 and STAT1 phosphorylation, inhibiting 52
IFNα-induced expression of 2’-5’-OAS in vector control cells and PLpro-expressing 53
cells. Overall results proved down-regulation of ERK1 by ubiquitin proteasomes and 54
suppression of interaction between ERK1 and STAT1 as Type I IFN antagonist 55
function of SARS-CoV PLpro.
56 57
Keywords: SARS coronavirus, papain-like protease, deubiquitination, interferon-α, 58
ERK1, STAT1 59
Introduction 61
Severe acute respiratory syndrome (SARS)-associated coronavirus 62
(SARS-CoV) is a novel pandemic virus causing highly contagious respiratorydisease 63
with approximately 10% mortality rate (Hsueh et al., 2004; Lee et al., 2003; Tsang et 64
al., 2003). Pathology entails bronchial epithelial denudation, loss of cilia, 65
multinucleated syncytial cells, squamous metaplasia and transendothelial migration of 66
monocytes/macrophages and neutrophils into lung tissue (Hsueh et al., 2004; Nicholls 67
et al., 2003). Hematological examination reveals lymphopenia, thrombocytopenia and 68
leukopenia (Wang et al., 2004b; Yan et al., 2004) accompanied by rapid elevation in 69
serum of inflammatory cytokines like IFN-gamma, IL-18, TGF-beta, IL-6, IP-10, 70
MCP-1, MIG, and IL-8, which stimulate recruitment of neutrophils, monocytes, and 71
immune responder cells like natural killer (NK), T, and B cells into lungs and other 72
organs (He et al., 2006; Huang et al., 2005; Wong et al., 2004).
73
SARS-CoV genome is an ~30 kbp positive-stranded RNA with a 5’ cap and 74
a 3’ poly(A) tract that contains 14 open reading frames (ORFs) (Marra et al., 2003;
75
Rota et al., 2003; Ziebuhr, 2004). The 5’ proximal and largest of these ORFs encodes 76
two large overlapping polyproteins replicase 1a and 1ab (~ 450 kDa and ~750 kDa, 77
respectively) processed to produce nonstructural (NS) proteins primarily involved in 78
RNA replication. Two specific embedded proteases, papain-like (PLpro) and 3C-like 79
(3CLpro), mediate processing of 1a and 1ab precursors into 16 NS proteins (termed 80
NS 1 through NS16).
81
PLpro, located within NS3, cleaves at NS1/2, NS2/3 and NS3/4 boundaries 82
using consensusmotif LXGG (Barretto et al., 2005; Lindner et al., 2005; Thiel et al., 83
2003), along with consensus cleavage sequence of cellular deubiquitinating enzymes.
84
Modeling and crystal structures reveal correlation between SARS-CoV PLpro and the 85
herpes virus-associated ubiquitin-specific protease (HAUSP), indicating potential 86
deubiquitinating activity (Ratia et al., 2006; Sulea et al., 2005) observed in in vitro 87
cleavage assays (Barretto et al., 2005; Lindner et al., 2005). Interestingly, one such in 88
vitro deubiquination assay measured the cleavage of ubiquitin-like protein, interferon 89
(IFN)-induced 15-kDa protein (ISG15), from an ISG15-fusion protein, suggesting 90
de-ISGylation by PLpro as a mechanism by which SARS-CoV inactivates 91
IFNα/β-induced innate immune response.
92
SARS-CoV infectiondoes not induce Type I IFNs in cell culture (Spiegel et 93
al., 2005). Recent reports reveal PLpro inhibitingthe phosphorylation of interferon 94
regulatory factor 3 (IRF-3) and Type I IFN synthesis (Devaraj et al., 2007) and 95
antagonizing both IRF-3 and NF-κB signaling pathways (Frieman et al., 2009). Still, 96
mechanisms of Type I IFN antagonism by which SARS-CoV PLpro does this remain 97
unclear. Type I interferons (IFNs, IFNα, IFNβ, and IFNω) mediate a wide range of 98
biological activities: antiviral activity, immune response, differentiation, cell growth, 99
apoptosis (Biron, 2001). IFN-α/β binds to common heterodimeric receptor composed 100
of IFN-α/β Receptor 1 (IFNAR1) and IFN-α/β Receptor 2 (IFNAR2), then activates 101
Janus kinase (JAK) family plus signal transducers and activators of transcription 102
(STATs) family (Tang et al., 2007). Phosphorylation of STAT1 at tyrosine 701 by 103
JAK1 is required for STAT1-STAT2 heterodimer formation and nuclear translocation 104
(Banninger & Reich, 2004). Phosphorylation of STAT1 at serine 727 by ERK1/2 and 105
p38 MAPK facilitates interaction of STAT1 with basal transcription machinery for full 106
expression of antiviral genes like Protein kinase R (PKR), 2’5’-oligoadenylate 107
synthetase (OAS), and IFN-stimulated gene 15 (ISG15) (Deb et al., 2003; Uddin et al., 108
2002). Currently, IFNα is also a widely used cytokine for treating human solid and 109
haematologic malignancies (Tagliaferri et al., 2005). IFNα-mediated anti-tumor effect 110
correlates with activation of JAK-STAT signaling pathway, resulting in up-regulation 111
of Fas/FasL and Jnk1/p38 stimulation signaling pathways. Escape mechanisms of 112
IFNα-mediated anti-tumor effect are likewise reported: e.g., EGF-mediated 113
Ras/Raf/ERK1-2–dependent pathway, Akt and NFkB-dependent pathways and 114
STAT3/PI3 K–mediated signaling (Tagliaferri et al., 2005). Some key regulators of 115
signal transduction―e.g., JAK1, STAT1, ERK1―are demonstrably modified by 116
ubiquitin conjugation (Malakhov et al., 2003; Zhimin & Tony, 2009), with over 100 117
ubiquitin-conjugated proteins encompassing diverse cellular pathways identified in 118
antiviral innate immune responses (Giannakopoulos et al., 2005; Zhao et al., 2005):
119
e.g., NF-κB-inducing kinase (NIK), critical regulator of noncanonical NF-κB pathway, 120
is ubiquitinated and degraded by RING finger E3 ligases (Varfolomeev et al., 2007).
121
With SARS-CoV PLpro as a deubiquitinating enzyme, this points to specifically 122
disrupting signal transduction of innate immune system against SARS-CoV infection.
123
Investigating possible effect of PLpro on the responses to type I IFNs is vital 124
to understanding SARS pathogenesis. This study first demonstrated stable expression 125
of SARS-CoV PLpro significantly inhibited IFNα-induced responses like ISRE- and 126
AP-1-driven promoter activation, gene expression of PKR, 2’-5’-OAS, IL-6 and IL-8, 127
and phosphorylation of STAT1 and c-Jun. Down-regulation of ERK1 was identified 128
by comparative proteomic analysis of PLpro-expressing vs. control cells with respect 129
to IFNα response, correlating with potential antagonistic mechanism of SARS-CoV 130
PLpro in response to IFNα. 131
132
Results 133
Expression of the SARS-CoV PLpro in human promonocytes 134
To characterize effect of SARS-CoV PLpro on the intracellular innate 135
immune response, human promonocyte HL-CZ cells were co-transfected with the 136
plasmid pSARS-CoV PLpro (expressing PLpro with HSV epitope tag) or empty 137
control vector and GFP reporter plasmid followed by two weeks of treatment with 138
G418 to select stably transfected cells. Expression of PLpro was detected by 139
immunofluorescent staining (Fig. 1A) and Western blotting (Fig. 1B), with 140
vector-derived HSV-tag found in both empty vector- and pSARS-CoV PLpro- 141
transfected cells and HSV-tag detected only in pSARS-CoV-PLpro-transfected 142
cells. Western blotting of transfected cells’ lysates with anti-HSV-tag antibodies 143
revealed a 60-kDa band in pSARS-CoV-PLpro- transfected cells (Fig. 1B), not in 144
empty vector-transfected cells.
145
To determine if expressed PLpro was active, proteolytic activity in cell 146
lysates was assayed by in-vitro trans-cleavage, with HRP containing LXGGmotif 147
recognized by PLpro as substrate. Fig. 1C shows significant reduction in HRP 148
enzyme activity in the reaction containing lysates of PLpro-expressing cells, not 149
in reaction with lysates from vector control cells. Lysates of PLpro-expressing 150
cells also exhibited time-dependent trans-cleavage activity. SARS-CoV PLpro 151
expressed in human promonocyte cells was thus enzymatically active.
152 153
Inhibition of PLpro on IFNα-induced ISRE- and AP-1-mediated activation 154
To test effect of SARS-CoV PLpro on ISRE-mediated responses to IFNα, 155
activity of ISRE-driven reporter and mRNA expression of ISRE-driven gene PKR in 156
empty vector controls and PLpro-expressing cells were examined by dual luciferase 157
reporter assay system (Fig. 2A) and quantitative real-time RT-PCR (Fig. 2B). Cells 158
were co-transfected with cis-reporter plasmid containing firefly luciferase under 159
control of the ISRE and an internal control reporter plasmid that constitutively 160
expressed renilla luciferase. After treatment with IFNα for 4 h, expression of firefly 161
luciferase was determined and normalized to renilla luciferase expression. Fig. 2A 162
plots vector control and PLpro-expressing cells’ dose-dependent transcriptional 163
activity of ISRE promoter by IFNα. ISRE promoter-driven luciferase activity in 164
PLpro-expressing cells was half that in vector control cells. The mRNA expression of 165
specific ISRE-driven gene PKR was analyzed in both types of cells in the absence or 166
presence of IFNα, using quantitative real-time RT-PCR assays (Fig. 2B). Induction of 167
PKR by IFNα was ~7 fold lower in PLpro expressing cells than in control vector 168
cells. Since endogenous PKR promoter contains not only ISRE element but also 169
kinase-conserved sequence (KCS) element for both basal and IFN-inducible PKR 170
promoter activity (Samuel, 2001), the other specific ISRE promoter-driven gene 171
2’-5’-OAS was further analyzed (Fig. 2C). Induction of 2’-5’-OAS by IFNα was 172
6-fold lower in PLpro-expressing cells than in vector controls. Results confirmed the 173
antagonism of IFNα−induced ISRE-mediated gene expression by PLpro.
174
Subsequently, effect of SARS-CoV PLpro on AP-1-mediated responses to 175
IFNα was tested (Fig. 3). Activity of AP-1 enhancer in response to IFNα was next 176
determined by transient transfection with plasmid vector containing luciferase under 177
control of the AP-1 enhancer. Fig. 3A shows luciferase activity significantly induced 178
in a dose-dependent manner in control vector cells by IFNα, but induction using the 179
same level of IFNα totally absent in PLpro-expressing cells. These results indicate 180
SARS-CoV PLpro mediated suppression AP-1-mediated promoter activity in 181
response to IFNα. Upon stimulation with IFNα, a 15-fold increase in IL-6 mRNA 182
was induced in vector control cells; no significant induction occurred in 183
PLpro-expressing cells (Fig. 3B). Since the AP-1 element was also required for the 184
IL-8 expression (Hoffmann et al., 2002), thus IL-8 mRNA levels in response to 185
IFNα were also measured (Fig. 3C). Levels of IL-8 mRNA were 3.5-fold higher in 186
both unstimulated and stimulated vector controls than in unstimulated and stimulated 187
PLpro-expressing cells (Fig. 3C), suggesting interference by PLpro with basal level 188
IL-8 mRNA transcription. AP-1 promoter activity and driven gene expression 189
indicated SARS-CoV PLpro as significantly inhibiting mRNA expression of 190
AP-1-mediated genes.
191 192
Down-regulation of IFNα-induced ERK1-mediated signaling by PLpro 193
For a global perspective mechanism of Type I IFN antagonism by 194
SARS-CoV PLpro, differential protein expression in vector control and 195
PLpro-expressing cells in the absence or presence of IFNα was analyzed by 196
two-dimensional electrophoresis (2-D) gel and nanoscale capillary liquid 197
chromatography/electrospray ionization Q-TOF MS to identify differentially 198
regulated proteins. In Fig 4A, down-regulated protein extracellular signal-regulated 199
kinase 1 (ERK1) and up-regulated ubiquitin-conjugating enzyme (UBC) E2-25K 200
appeared in 2D gels of IFNα-treated PLpro-expressing cells, and then identified by 201
trypsin digestion and NanoLC Trap Q-TOF MS analysis. ERK1 showed a Mascot 202
score of 109, sequence coverage of 14%, and 2 matched peptides; UBC E2-25K 203
showed a Mascot score of 248, sequence coverage of 59%, and 4 matched peptides.
204
Peptide peaks from Q-TOF MS analysis from two representative spots of ERK1 and 205
UBC E2-25K (Figs. 4B-4C, respectively). ERK1 in particular is reported in several 206
biological pathways (mitogen-activated protein kinase kinase, cytokine-mediated 207
inflammation, IFN signaling pathways) and thus could play an important role in the 208
mechanism of IFNα antagonism by PLpro.
209
Up-regulation of UBC E2-25K of ubiquitin proteasome pathways by PLpro 210
Quantitative RT-PCR was employed to determine expression levels of ERK1 211
and UBC E2-25K in PLpro-expressing and vector control cells in the absence or 212
presence of IFNα (Fig. 5). Amount of ERK1 mRNA showed no difference between 213
vector control and PLpro-expressing cells, whether treated with IFNα or not (Fig. 5A).
214
Relative level of UBC E2-25K mRNA in PLpro-expressing cells was markedly higher 215
than that in vector controls, with or without IFNα treatment (Fig. 5B), proving that 216
SARS-CoV PLpro activates the ubiquitin-proteasome system in human promonocyte 217
cells. To compare ERK1 protein levels in vector control and PLpro-expressing cells in 218
the presence or absence of IFNα, ERK1 and ERK2 were measured by Western blots 219
with anti-p44/p42 (ERK1/2) monoclonal antibody (Fig. 6A). Western blotting showed 220
42-kDa ERK2 protein levels roughly similar in vector control and PLpro-expressing 221
cells, whereas the protein level of 44-kDa ERK1 in PLpro-expressing cells was near 222
50% of that in controls (determined by densitometry normalized to β-actin protein 223
control in each sample) (Fig. 6A, Lanes 1-2). IFNα treatment caused time-dependent 224
reduction of ERK1, but not ERK2, in PLpro-expressing cells (Fig. 6A, Lanes 4 and 6).
225
Results confirmed data of 2-D/MALDI TOF MS, which showed definite reduction of 226
ERK1 in PLpro-expressing cells in response to IFNα.
227
Since PLpro-expressing cells have no difference in mRNA amount, but a 228
significantly reduction of ERK1 protein levels by IFNα, we suggest that up-regulation 229
of UBC E2-25k in PLpro-expressing cells could increase ubiquitination on ERK1, 230
enhancing ERK1 degradation by IFNα treatment. To test the hypothesis, ERK1 231
immunoprecipition followed by Western blot probed with anti-ubiquitin antibodies 232
was conducted in the absence or presence of IFNα (Fig 6B), revealing that ERK1 233
conjugated with different sizes of poly-ubiquitin chains: i.e., molecular sizes of 52, 60, 234
68, 76, and 84 kDa. Higher level of ERK1 ubiquitination was found in 235
PLpro-expressing cells (Fig. 6B, Lane 2) than in vector control cells (Fig. 6B, Lane 1).
236
Moreover, IFNα treatment significantly reduced the level of ERK1 ubiquitination in 237
PLpro-expressing cells (Fig. 6B, Lane 4), not in vector controls (Fig. 6B, Lane 3).
238
To test correlation between up-regulation of unbiquitin proteasome activity 239
and down-regulation of ERK1 in PLpro-expressing cells, proteasome inhibitor 240
MG-132 was added to analyze changes of ERK1 and ERK2 using Western blot assays 241
with anti-p44/p42 (ERK1/2) monoclonal antibody (Fig. 6C). Treatment with both 242
IFNα and proteasome inhibitor MG-132 caused time-dependent increases of ERK1 243
and ERK2, in PLpro-expressing cells (Fig 6C, Lanes 2, 4, 6, and 8). The higher 244
expression level of ERK2 than ERK1 was consistently observed in vector control and 245
PLpro-expressing cells in responses to treatment with/without both IFNα and 246
proteasome inhibitor MG-132. The increase of ERK1 level in PLpro-expressing cells 247
correlated with treatment of proteasome inhibitor MG-132, being not compensated by 248
ERK2. After 1 h treatment with both IFNα and MG-132, overall amount of ERK1 in 249
PLpro-expressing cells was equal to that in vector control cells (Fig 6C, Lanes 7 and 250
8). Results indicate proteasome inhibitor MG-132 blocking escape of IFNα-induced 251
response by ERK1 degradation in PLpro-expressing cells, along with SARS-CoV 252
PLpro enhancing ERK1 degradation by up-regulating ubiquitin proteasome pathways 253
in response to IFNα, being associated with inhibiting IFNα-induced ISRE- and AP-1 254
promoter activation and IFNα-stimulated gene expression.
255 256
Inhibition of ubiquitin proteasome activity restored activation of IFNα-induced 257
ERK-mediating signaling in PLpro-expressing cells 258
To examine effects of unbiquitin proteasome up-regulation on 259
ERK1-mediated signaling, proteasome inhibitor MG-132 was added to analyze 260
changes of ERK1-mediated signaling pathway. Phosphorylation of ERK1, STAT1 and 261
c-Jun in PLpro-expressing cells and vector control cells was subsequently analyzed by 262
Western blots with phosphorylation site-specific antibodies (Fig. 7). IFNα treatment 263
caused time-dependent ERK1 phosphorylation in vector controls (Fig. 7A, Lanes 1, 3, 264
5, and 7), but only a transient period of ERK1 phosphorylation in PLpro-expressing 265
cells (Fig. 7A, Lane 4), probably due to lower ERK1 protein levels via degradation by 266
ubiquitin-proteasome pathway in PLpro-expressing cells following IFNα treatment 267
(Fig. 6). Consistent with this hypothesis, treatment with both IFNα and proteasome 268
inhibitor MG-132 restored IFNα-induced activation of ERK1 in a time-dependent 269
manner in PLpro-expressing cells (Fig. 7B, Lanes 2, 4, 6, and 8). Treatment with 270
IFNα or both IFNα and the proteasome inhibitor MG-132 had no detectable band of 271
phospho-ERK2 in vector control and PLpro-expressing cells. Subsequently, PLpro 272
expression suppressed phosphorylation of STAT1 at Tyr701 and Ser727 sites in 273
resting cells and in response to IFNα treatment (Fig. 7C, Lanes 4, 6, and 8). Treatment 274
with proteasome inhibitor MG-132 also significantly increased phosphorylation of 275
STAT1 at Tyr701 and Ser727 sites in PLpro-expressing cells induced with IFNα (Fig.
276
7D, Lanes 4, 6, and 8). Moreover, phosphorylation of transcriptional factor c-Jun was 277
assessed to find level of c-Jun phosphorylation similar in both types of cells. Yet IFNα 278
treatment reduced c-Jun phosphorylation, meanwhile treatment with both IFNα and 279
MG-132 also significantly increased c-Jun phosphorylation in PLpro-expressing cells 280
(Figs. 7C and 7D, Lanes 4, 6, and 8). As expected, if PLpro-induced degradation of 281
ERK1 suppresses STAT1 and c-Jun activation, inhibition of ubiquitin proteasome 282
function with MG132 heightened IFNα-induced activation of ERK1-mediated 283
signaling in PLpro-expressing cells.
284
285
Correlation of ERK1 phosphorylation with STAT1 signaling pathways 286
To confirm effect of ERK1 phosphorylation on STAT1 signaling, inhibition 287
of PD098059 (an ERK1/2 inhibitor) on ERK1 and STAT1 phosphorylation was 288
analyzed by Western blotting (Fig. 8). PD098059 treatment had inhibitory effects on 289
IFNα-induced ERK1 phosphorylation in vector control cells and PLpro-expressing 290
cells (Fig. 8A, Lanes 5-7; Fig. 8B, Lanes 5-7). Importantly, PD098059 treatment also 291
manifests inhibitory effects on STAT1 phosphorylation at Ser727, but not Tyr701 in 292
vector control cells and PLpro-expressing cells in response to IFNα treatment (Fig.
293
8A, Lanes 5-7; Fig. 8B, Lanes 5-7). In addition, effects of PD098059 treatment on 294
IFNα-induced ISRE promoter-driven gene expression were further investigated using 295
real time RT-PCR (Supplemental Fig. 1). PD098059 treatment starkly reduced 296
IFNα-induced expression of 2’-5’-OAS in vector control and PLpro-expressing cells 297
(Supplemental Fig. 1). Results confirmed a link between ERK1 activation and 298
STAT1 signaling as the antagonism of IFNα−induced ISRE-mediated gene 299
expression by PLpro.
300 301
Discussion 302
SARS-CoV does not induce type I IFN in cell culture, which may be crucial 303
to pathogenesis of this virus. This study focused on one SARS-CoV protein, PLpro 304
protease, earlier reported to have antagonistic activity in innate immune responses 305
including synthesis of IFNs and cytokines (Devaraj et al., 2007; Frieman et al., 2009).
306
We first demonstrated stable SARS-CoV PLpro expression in human promonocyte 307
cells as well as inhibition of IFNα-induced ISRE- and AP-1-driven promoter activity 308
and reduction of IFN-stimulated gene expression (Figs. 2-3). Results concurred with 309
previous findings: SARS-CoV PLpro protein inhibited activity of IFNβ, ISRE and 310
NF-κB promoters induced by polyI:C (Devaraj et al., 2007; Frieman et al., 2009).
311
The antagonistic mechanism of SARS-CoV PLpro on these activities is controversial 312
(Devaraj et al., 2007; Frieman et al., 2009). Devaraj and colleagues demonstrated 313
PLpro interacting with IRF-3, blocking phosphorylation and nuclear translocation of 314
IRF-3 and disrupting activation of Type I IFN responses (Devaraj et al., 2007).
315
Frieman and colleagues found PLpro not directly binding with IRF-3 or inhibiting in 316
vitro phosphorylation of IRF-3 (Frieman et al., 2009).
317
This study used proteomic approach to detect changes in protein expression 318
in PLpro-expressing cells in the presence or absence of IFNα (Fig. 4). PLpro 319
expression in human promonocyte cells stimulated mRNA expression of UBC 320
E2-25K (Fig. 5B), which could support increase of protein level of UBC E2-25K in 321
2-D gels (Fig. 4). PLpro expression caused 50% decrease of ERK1, but not ERK2, in 322
PLpro-expressing cells compared to vector controls (Fig 6A), being associated with 323
ubiquitin-dependent proteosomal degradation of ERK1, as confirmed by 324
poly-ubiquitination of ERK1 and treatment with proteosome inhibitor MG132 (Figs.
325
6B-6C). IFNα treatment enhanced time-dependent manner of ERK1 down-regulation, 326
but proteosome inhibitor MG132 time-dependently restored IFNα-enhanced 327
degradation of ERK1 in PLpro-expressing cells, but not vector controls (Figs. 6A 328
and 6C). With ERK1/2 signaling regulated by ubiquitin-proteasome system via 329
degradation of ERK1/2 and the upstream MEKK1 by ubiquitination (Laine & Ronai, 330
2005; Lu et al., 2002), those reports led us to identify ERK1 ubiquitination level in 331
vector control and PLpro-expressing cells with or without IFNα treatment (Fig. 6B).
332
Interestingly, PLpro expression significantly increased ERK1 ubiquitination with 333
poly-ubiquitin chains compared to vector control cells (Fig. 6B, Lanes 1-2), while 334
IFNα treatment decreased ubiquitinated levels and protein amounts of ERK1 in 335
PLpro-expressing cells, not in vector control cells (Fig. 6B, Lanes 3-4). Treatment 336
with proteasome inhibitor MG132 restored protein amounts of ERK1 (Fig. 6C) and 337
IFNα-induced activation of ERK1-mdiated signaling in PLpro-expressing cells (Fig 338
8), in concordance with prior studies: i.e., ERK1/2 signaling regulated by 339
ubiquitin-proteasome system via degradation of ERK1/2 and upstream MEKK1 by 340
ubiquitination (Laine & Ronai, 2005; Lu et al., 2002). Proteomic analysis identified 341
down-regulation of ERK1 that was ubiquitinated and degraded by up-regulation of 342
ubiquitin proteasome pathways in PLpro-expressing cells, being responsible for the 343
mechanism of IFNα antagonism by SARS-CoV PLpro.
344
The treatment with proteasome inhibitor MG132 reversed this inhibition of 345
IFNα-induced ERK1-mediated signaling by PLpro (Fig. 7), indicating a significant 346
correlation between ERK1 and STAT1 in PLpro-expressing cells in response to IFNα. 347
Results concurred with prior studies, with phosphorylation at Serine 727 of STAT1 348
by active ERK1 involved in IFNα/β-induced response (Wang et al., 2004a) and IFNγ 349
inflammatory response (Lombardi et al., 2008; Matsumoto et al., 2005). In addition, 350
down-regulation of ERK1 in PLpro-expression cells correlated with suppression of 351
AP-1-driven luciferase activity, IL-6 and IL-8 mRNA expression and c-Jun 352
phosphorylation in responses to IFNβ(Figs. 3 and 7). Importantly, we confirmed the 353
correlation of ERK1 and STAT1 signaling pathways by treatment of PD098059 (an 354
ERK1/2 inhibitor) (Fig. 8). PD098059 treatment inhibited IFNα-induced ERK1 and 355
STAT1 phosphorylation in vector control and PLpro-expressing cells, as well as 356
IFNα-induced expression of 2’-5’-OAS in vector control and PLpro-expressing cells 357
(Supplemental Fig. 1). In addition, the other ERK1/2 inhibitor U0126 was used to 358
test the correlation between ERK1/2 and STAT1. ERK1/2 inhibitor U0126 359
significantly inhibited IFN-alpha-induced phosphorylation of STAT1 at Ser727 in 360
vector control cells and PLpro-expressing cells (Supplemental Fig. 2).
361
ERK1/2-mediated signaling proves elemental in EGF-induced survival response to 362
antagonize IFNα-induced apoptosis of cancer cells (Caraglia et al., 2003).
363
Down-regulation of ERK1-mediated signaling by PLpro might thus be considered in 364
escape mechanism of SARS-CoV against Type I IFNs. Activation of ERK1-mediated 365
signaling may improve innate immune response against SARS-CoV, being 366
alternative targets for development of SARS therapy.
367
We also demonstrated reduction of ERK1 protein level in human 368
promonocyte cells 24 hours post infection with human coronavirus NL63 369
(HCoV-NL63) and reversion of ERK1 protein level in HCoV-NL63-infected cells 370
after a 24-hour incubation of IFNα and proteasome inhibitor MG132 (Supplemental 371
Fig. 3). In addition, the reduction of IFNα-induced phosphorylation of both ERK1 372
and STAT1 at Ser727 was confirmed in human lung adenocarcinoma epithelial A549 373
cells expressing SARS-CoV PLpro compared to vector control (Supplemental Fig. 4).
374
Surprisingly, ERK2 that had the consistently higher expression level than ERK1 in 375
vector control and PLpro-expressing cells showed fewer amounts of protein level and 376
IFNα-induced phosphorylation in PLpro-expressing cells than vector control cells 377
(Figs. 6A, 7A, and 8, Supplemental Fig. 4). The treatment with proteasome inhibitor 378
MG132 reversed the amounts of ERK2 protein and the inhibition of IFNα-induced 379
ERK2 phosphorylation in PLpro-expressing cells (Figs. 6C and 7B). Besides ERK1, 380
ERK2 might be involved in Type I IFN antagonism by SARS-CoV PLpro. ERK1 and 381
ERK2 have approximately 85% of amino acid identity co-expressed in virtually all 382
tissues but with remarkably variable relative abundance, ERK2 as the predominant 383
isoform in brain and hematopoietic cells (Milella et al., 2003; Pages & Pouyssegur, 384
2004). Recent evidence suggests possible quantitative difference in ERK1 and ERK2 385
dynamics that could have a significant role in their regulation. Ectopic expression of 386
ERK1, albeit not ERK2, attenuates Ras-dependent tumor formation in nude mice 387
(Vantaggiato et al., 2006). The properties of their cytoplasmic-nuclear trafficking 388
showed ERK1 shuttles between nucleus and cytoplasm at a much slower rate than 389
ERK2, correlating with reduced capability of ERK1 to carry proliferative signals to 390
the nucleus (Marchi et al., 2008). Constitutive activation of ERK2, but not ERK1, is 391
critical for the acquired resistance to Imatinib Mesylate in chronic myelogenous 392
leukemia management (Aceves-Luquero et al., 2009). In addition to cancers, Ebola 393
virus envelope glycoprotein reduced phosphorylation and kinase activity of ERK2, 394
but not ERK1, correlating with induction of cell death (Zampieri et al., 2007).
395
Vaccinia virus M2L protein blocks ERK2 phosphorylation, inhibiting virus-induced 396
NF-κB activation (Gedey et al., 2006). Type I IFN antagonism of SARS-CoV PLpro 397
via ERK1 down-regulation might thus be a unique mechanism useful in developing 398
therapeutic agents against SARS-CoV infection.
399
In conclusion, stable SARS-CoV PLpro expression significantly suppressed 400
IFNα-induced responses. Up-regulation of ubiquitin-proteasome pathway by 401
SARS-CoV PLpro correlated with increase of ERK1 ubiquitination. IFNα treatment 402
elicited ERK1 degradation, then down-regulated ERK1-mediated signaling in 403
PLpro-expressing cells, resulting in negative regulation of STAT1 and AP-1 signaling 404
pathways. Importantly, inhibition of ubiquitin proteasome function with MG132 405
restored IFNα-induced phosphorylation of ERK1, STAT1, and c-Jun, all suppressed 406
by SARS-CoV PLpro. PD098059 treatment confirmed linkage between ERK1 407
activation and STAT1 signaling pathways as Type I IFN antagonism by PLpro.
408
Moreover, the study may provide novel insight into the molecular mechanism of IFN 409
antagonism by SARS CoV PLpro.
410 411
Materials and methods 412
Cell culture and transfection 413
The SARS-CoV PLpro gene, located between nucleotides 4507-5840 of the 414
SARS-CoV TW1 strain genome (GenBank Accession No. AY291451), was 415
amplified by RT-PCR from genome RNA template, using primers 5′-CTCCGAAT 416
TCAACTCTCTAAATGAGCCGCTTGTC-3′ and 5′-GAGGCTCGAGATCCTCTGG 417
GTCTTCAGGAGCGAGTTCTGGCTGTACGACACAGGCTTGATGGTTGTAGT 418
G-3′. Forward primer contained EcoRI restriction site; reverse primer included an 419
XhoI restriction site and HSV epitope tag. Amplified RT-PCR product was cloned 420
into pcDNA3.1/His C vector (Invitrogen), resulting construct named pSARS-CoV 421
PLpro. The pSARS-CoV PLpro (4.5 μg) plus indicator vector pEGFP-N1 (0.5 μg) 422
(Clontech) or pcDNA3.1 empty vector plus pEGFP-N1 were transfected into HL-CZ 423
cells (human promonocyte cell line) with GenePorter reagent. As per manufacturer’s 424
direction (Gene Therapy Systems, San Diego, CA), transfected cells were incubated 425
for 5 hours with a mixture of plasmid DNA and GenePorter reagent, then maintained 426
in RPMI 1640 medium containing 20% bovine serum (FBS). For the selection of the 427
stably transfected cell line, cells were incubated with RPMI 1640 medium containing 428
10 % FBS and 800 µg/ml of G418. PLpro expression was detected by Western 429
blotting of transfected cell lysates, using anti-HSV Tag mAb (Novagen) as a probe.
430 431
In vitro trans-cleavage activity of SARS-CoV PLpro 432
The protease activity in SARS-CoV PLpro-transfected cellswas determined by 433
spectrophotometrically following digestion of substrate horseradish peroxidase (HRP) 434
containing the LXGGmotif (Sigma). 150 μl of transfected cell lysates were added to 435
150 μl of substrate reagent containing 0.01 μg/ml HRP in 50 mM Tris-HCl. After 1-, 436
2-, 3-, and 4-h incubation at 37℃, reaction mixtures were added to a 96-well plate and 437
non-digested HRP activity measured by adding chromogen solution containing 438
2,2’-azino-di-3- ethylbenzthiazoline-6-sulfonate (ABTS) and hydrogen peroxide.
439
Relative trans-cleavage activity was calculated as 1 – (A405PLpro)/(A405no PLpro).
440 441
Transient transfections of cis-reporter plasmids for signaling pathway assays 442
Plasmid pISRE-Luc cis-reporter was purchased from Stratagene. SARS-CoV 443
PLpro-expressing and empty vector control cells were transfected with cis-reporter 444
plasmid indicated, plus internal control reporter pRluc-C1 (BioSignal Packard) using 445
GenePorter reagent. After 4 h incubation with or without IFNα2 (Hoffmann-La 446
Roche), activity of experimental firefly luciferase and control renilla luciferase was 447
gauged by dual Luciferase Reporter Assay System (Promega) and TROPIX TR-717 448
Luminometer (Applied Biosystems) described by Lin et al. (Lin et al., 2008).
449 450
2-DE and protein spot analysis 451
For two-dimensional gel electrophoresis, empty vector control cells and 452
PLpro-expressing cells incubated for 3 days in the presence or absence of 3000 U/ml 453
IFNα were harvested, washed twice with ice-cold phosphate-buffered saline, and then 454
extracted with lysis buffer containing 8 M urea, 4% CHAPS, 2% pH 3-10 non-linear 455
(NL) IPG buffer (GE Healthcare), plus Complete, Mini, EDTA-free protease inhibitor 456
mixture (Roche). After 3 h incubation at 4℃, cell lysates were centrifuged for 15 min 457
at 16000 g. Protein concentration of resulting supernatants was gauged with Bio-Rad 458
Protein Assay (Bio-Rad, Hercules, CA, USA), 100 μg of protein sample diluted with 459
350 μl of rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer pH 3-10 NL, 18 460
mM dithiothreilol, 0.002% bromophenol blue), then applied to nonlinear Immobiline 461
DryStrips (17 cm, pH 3-10; GE Healthcare). First-dimensional isoelectric focus and 462
second-dimensional electrophoresis were detailed in Lai et al. (2007), as was in-gel 463
digestion to recover peptides from gel spots for nanoelectrospray mass spectrometry.
464 465
Nanoelectrospray mass spectrometry, data interpretation and database search 466
Proteins in spots of interest were identified using an Ultimate capillary LC 467
system (LC Packings, Amsterdam, The Netherlands) coupled to a QSTARXL 468
quadrupole-time of flight (Q-TOF) mass spectrometer (Applied Biosystem/MDS 469
Sciex, Foster City, CA, USA). The nanoelectrospray mass spectrometry and database 470
search were described previously (Lai et al., 2007). Protein function and subcellular 471
location were annotated by Swiss-Prot (http://us.expasy.org/sprot/) and proteins 472
categorized according to their biological process and pathway, using thePANTHER 473
Classification system (http://www.pantherdb.org) described in prior studies (Lai et al., 474
2007; Varfolomeev et al., 2007; Wang et al., 2004a; Wang et al., 2004b).
475 476
Western blotting and immunoprecipitation assays 477
To determine protein expression, lysates of PLpro-expressing cells and 478
empty vector control cells incubated for 1 day in the presence or absence of 3000 479
U/ml IFNα were mixed 1:1 with 2X SDS-PAGE sample buffer without 480
2-mercaptoethanol and boiled for 10 min. Proteins in the lysates were resolved by 481
SDS-PAGE and transferred to nitrocellulose. Resulting blots were blocked with 482
5% skim milk, then reacted with appropriately diluted antibodies, including rabbit 483
anti-STAT 1 (Cell Signaling), rabbit anti-phospho STAT 1 (Ser 727) (Abcam), 484
rabbit anti-phospho STAT 1 (Tyr 701) (Abcam), anti-ERK1/2 mAb (Cell 485
Signaling), anti-phospho-ERK1/2 mAb (Cell Signaling), rabbit anti-c-Jun 486
(Abcam), rabbit anti-phospho c-Jun (Abacm), and anti-ubiquitin mAb (Zymed).
487
Immune complexes were detected with horseradishperoxidase-conjugated goat 488
anti-mouse or anti-rabbit IgG antibodies, followed by enhanced 489
chemiluminescence detection (Amersham Pharmacia Biotech). To detect 490
ubiquitination of ERK1, cell lysates were harvested and incubated with 491
anti-ERK1 antibody for 4 h at 4℃, followed by addition of protein A-Sepharose 492
beads and additional 2 h of incubation. After collection by centrifugation, pellets 493
were washed four times with NET buffer (150 mM NaCl, 0.1 mM EDTA, 30 mM 494
Tris-HCl, pH 7.4); immunoprecipitated proteins were dissolved in 2X SDS-PAGE 495
sample buffer without 2-mercaptoethanol and boiled for 10 min. Proteins were 496
resolved by SDS-PAGE and transferred to nitrocellulose. Resulting blots were 497
blocked with 5% skim milk and then probed with rabbit anti-ERK1 (Zymed) and 498
anti-ubiquitin mAb (Zymed) followed by enhanced chemiluminescence detection.
499 500
Quantification of IFNβ mRNA using real time RT-PCR 501
Total RNA was isolated from PLpro-expressing cells and empty vector 502
control cells incubated for 4 hrs in the presence or absence of 3000 U/ml IFNα, using 503
PureLink Micro-to-Midi Total RNA Purification System Kit (Invitrogen). cDNA was 504
synthesized from 1000 ng of total RNA, using oligonucleotide dT primer and 505
SuperScript III reverse transcriptase kit (Invitrogen). To gauge expression in response 506
to IFNα, a two-step RT-PCR using SYBR Green I was used. Oligonucleotide primer 507
pairs were (1) forward primer 5’-CAACCAGCGGTTGACTTTTT-3’ and reverse 508
primer 5’-ATCCAGGAAGGCAAACTGAA-3’ for human PKR, (2) forward primer 509
5’-GATGTGCTGCCTGCCTTT-3’ and reverse primer 5’- TTGGGGGTTAGGTTT 510
ATAGCTG-3’ for human 2’-5’-OAS, (3) forward primer 5’-GATGGATGCTTCCAAT 511
CTGGAT-3’ and reverse primer 5’- AGTTCTCCATAGAGAACAACATA-3’ for 512
human IL-6, (4) forward primer 5’- CGA TGTCAGTGCATAAAGACA -3’ and 513
reverse primer 5’- TGAATTCTCAGCCCT CTTCAAAAA-3’ for human IL-8, (5) 514
forward primer 5’-CTTCCCTGGCAAGCACTACC-3’ and reverse primer 515
5’-GTTTCGGGCTTCATGTTGA-3’ for human ERK1, and (6) forward primer 516
5’-GCAATGACTCTCCGCACGG-3’ and reverse primer 5’-TCTGTTGCAGTCTCT 517
ACATCCC-3’ for human UBC E2-25K. In addition, glyceraldehyde-3-phosphate 518
dehydrogenase (GAPDH) mRNA, a housekeeping gene, was measured using 519
5’-AGCCACATCGCTCAGACAC-3’ and 5’-GCCCCA ATACGACCAAATCC-3’ as 520
forward and reverse primers. Real-time PCR reaction mixture contained 2.5 μl of 521
cDNA (reverse transcription mixture), 200 nM of each primer in SYBR Green I 522
master mix (LightCycler TaqMAn Master, Roche Diagnostics). PCR was performed 523
with amplification protocol consisting of 1 cycle at 50℃ for 2 min, 1 cycle at 95℃for 524
10 min, 45 cycles at 95℃ for 15 sec, and 60℃ for 1 min. Amplification and detection 525
of specific products were conducted in ABI PRISM 7700 sequence detection system 526
(PE Applied Biosystems). Relative changes in mRNA level of indicated genes were 527
normalized relative to GAPDH mRNA.
528 529
Statistical analysis 530
Student's t-test or Chi-square test analyzed all data. Statistical significance 531
between vector-control cells and PLpro-expressing cells was noted at p < 0.05.
532 533
Acknowledgment 534
We would like to thank the National Science Council (Taiwan) and China 535
Medical University for financial support (NSC96-2320-B-039-008-MY3 and 536
CMU98-CT-22, CMU98-D-S-05, CMU98-P-03).
537 538
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Figure captions
728
Fig. 1. Expression of SARS-CoV PLpro in human promonocyte HL-CZ cells.
729
Cells transfected with pcDNA3.1 (control vector) plus pEGFP-N1 or 730
pSARS-CoV-PLpro plus pEGFP-N1 were selected by a 2-week incubation with 731
G418. The HSV-tag fusion protein was detected using immunofluorescence 732
staining of anti-HSV tag antibody and rhodamineconjugated antimouse IgG 733
antibody (A). Lysates from cells transfected with pcDNA3.1 plus pEGFP-N1 734
(lane 1) or pSARS-CoV-PLpro plus pEGFP-N1 (lane 2) were analyzed by 10%
735
SDS-PAGE prior to blotting (B). The blot’s upper half of was probed with 736
anti-HSV antibody, the lower with anti-β actin antibody as internal control.
737
Trans-cleavage activity of SARS-CoV PLpro in transfected cell lysates was 738
further analyzed (C). Following incubation of lysates from 106 PLpro-expressing 739
cells and control vector cells with substrate HRP, residual HRP activity was 740
measured as a mean of 3 independent experiments; error bars show standard error 741
of the mean 742
743
Fig. 2. Effect of PLpro on ISRE mediated gene expression in response to IFNα. 744
(A) Vector control cells and PLro-expressing cells were transiently co-transfected 745
with reporter plasmid containing firefly luciferase under control of the ISRE and 746
an internal control reporter pRluc-C1 that constitutively expressed Renilla 747
luciferase. After 4-hour IFNαtreatment, firefly luciferase and renilla luciferase 748
were measured and firefly luciferase activity normalized to Renilla luciferase 749
activity, as reported. Each bar is the mean of 3 independent experiments; error bar 750
is standard error of the mean. The mRNA expressions of ISRE-driven gene PKR 751
(B) and 2’-5’-OAS (C) in vector control cells and SARS PLpro-expressing cells 752
untreated or treated was measured by quantitative real time PCR. Relative fold 753
levels of PKR or 2’-5’-OAS mRNA level appear as ratio of PKR or 2’-5’-OAS 754
mRNA/GAPDHmRNA. Each bar graph is the mean of 3 independent experiments;
755
error bars represent standard error of the mean.
756 757
Fig. 3. Effect of PLpro on AP-1 mediated gene expression in response to IFNα. (A) 758
Vector control and PLpro-expressing cells were transiently co-transfected with 759
reporter plasmid containing AP-1-driven firefly luciferase and an internal control 760
reporter pRluc-C1 that constitutively expressed renilla luciferase. After 4-hour 761
treatment with IFNα, AP-1-driven firefly luciferase and renilla luciferase were 762
measured and firefly luciferase activity normalized to renilla luciferase activity is 763
reported. Each bar is the mean of 3 independent experiments; error bar is standard 764
error of the mean. In addition, the mRNA expressions of AP-1-driven genes IL-6 765
(B) and IL-8 (C) in vector control cells and SARS PLpro-expressing cells 766
untreated or treated was measured by quantitative real time PCR. Relative fold 767
levels of IL-6 or IL-8 mRNA level are presented as the ratio of IL-6 or IL-8 768
mRNA/GAPDHmRNA. Each bar on the graph is the mean of 3 independent 769
experiments; error bars represent standard error of the mean.
770 771
Fig. 4. Effect of SARS-CoV PLpro on protein profiles of vector control cells and 772
PLpro-expressing cells in response to IFNα. 100 μg of total protein from control 773
vector cells in the absence or presence of IFNα or PLpro-expressing cells in the 774
absence or presence of IFNα was resolved by 2-dimensional electrophoresis. (A) 775
Enlarged images of two-dimensional gel electrophoresis of protein expression in 776
PLpro-expressing cells and vector control cells in response to IFNα treatment. (B) 777
Nanoelectrospray mass spectrum of triply charged ion m/z 1514.77 for ERK1 is 778
shown; ITVEEALAHPYLEQYYDPTDEPVAEEPFTFAMoxELDDLPK amino 779
acid sequence was determined from mass differences in y- and b-fragment ions 780
series and matched residues 319-357 of ERK1 (mitogen-activated protein kinase 781
3). (C) Nanoelectrospray mass spectrum of the doubly charged ion m/z 725.41 for 782
UBC E2-25k is shown. Amino acid sequence VDLVDENFTELR was determined 783
from mass differences in y- and b-fragment ions series and matched residues 784
29-40 of ubiquitin-conjugating enzyme E2-25k. *Only y- and b-fragment ions are 785
labeled in the spectrum.
786 787
Fig. 5. Analysis of mRNA levels of ERK1 and UBC E2-25K in vector control cells 788
and PLpro-expressing cells. Total RNA was extracted from vector control cells 789
and PLpro-expressing cells treated with or without IFNα (3000U/ml) for 4 hrs and 790
relative mRNA levels of ERK1 (A) and UBC E2-25K (B) were measured by 791
quantitative real time PCR. The relative fold levels of ERK1 and UBC E2-25K 792
mRNA were presented as the ratio of indicated mRNA/GAPDHmRNA. Each bar 793
on the graph is the mean of 3 independent experiments and the error bars represent 794
the standard error of the mean.
795 796
Fig. 6. Protein amount and ubiquitination level of ERK1 in vector control cells 797
and PLpro-expressing cells. (A) Vector control cells and PLpro-expressing cells 798
were treated with IFNα (3000U/ml) for 30 or 60 minutes. Cell lysates were 799
Western blotted and probed with anti-ERK1/2 or anti-β-actin antibody as an 800
internal control. (B) Vector control cells and PLpro-expressing cells were treated 801
with or without IFNα (3000U/ml) for 60 minutes. Cell lysates were also 802
immunoprecipitated with anti-ERK1 mAb, followed by Western blotting probed 803
with either anti-ubiquitin or anti-ERK1 antibody. (C) Vector control cells and 804
PLpro-expressing cells were treated with IFNα and the proteosome inhibitor 805
MG132 (20μM) for 10, 30, or 60 minutes. Cell lysates were Western blotted and 806
probed with anti-ERK1/2 or anti-β-actin antibody as an internal control.
807 808
Fig. 7. Effect of proteasome inhibitor MG132 on IFNα-induced phosphorylation 809
of ERK1, STAT1 and c-Jun in vector control cells and PLpro-expressing cells.
810
Vector control cells and PLpro-expressing cells were treated with IFNα (3000U/ml) 811
(A), or IFNα and proteasome inhibitor MG132 (20μM) (B) for 10, 30 or 60 812
minutes. Cell lysates were subjected to Western blotting probed with 813
anti-phospho-ERK1/2, anti-ERK1/2 anti-phospho-STAT1 (Tyr701), 814
anti-phospho-STAT1 (Ser727), anti-STAT1, anti-phospho-c-Jun or anti-c-Jun 815
antibodies. Relevant protein of the blot was probed with anti-β actin antibodies as 816
an internal control.
817 818
Fig. 8. Effect of PD098059 treatment on IFNα-induced phosphorylation of ERK1 819
and STAT1 in vector control cells and PLpro-expressing cells. Vector control 820
cells and PLpro-expressing cells were treated with IFNα (A), or IFNα and 821
PD098059 (B) for 10, 30 or 60 minutes. Cell lysates were subjected to Western 822
blotting probed with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT1 823
(Tyr701), anti-phospho-STAT1 (Ser727) or anti-STAT1 antibodies. Relevant 824
protein of the blot was probed with anti-β actin antibodies as an internal control.
825
