1 1 2 3 proteasome inhibition in vivo promotes survival in a lethal
TRANSCRIPT
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Proteasome inhibition in vivo promotes survival in a lethal murine model of SARS 4
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Xue-Zhong Ma1, Agata Bartczak
1, Jianhua Zhang
1, Ramzi Khattar
1, Limin Chen
2, Ming 6
Feng Liu6, Aled Edwards
2, Gary Levy
1,3, Ian D. McGilvray*,
1,4 7
8
From the 1 Multi-organ Transplant Program, Toronto General Research Institute, 9
University Health Network, University of Toronto, Toronto, ON, Canada; 2 Best 10
Department of Medical Research, University of Toronto, 3 Department of Medicine, 11
University of Toronto, and the, 4 Department of Surgery, University of Toronto 12
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Running title: Proteasome inhibition and MHV-1 14
Keywords: proteasome, coronavirus, macrophage, pneumonitis, SARS 15
Word count: Abstract (221); Text (5,280) 16
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Max Bell Research Institute 18
200 Elizabeth St, 19
Toronto, ON, M5G-2C4 20
Canada 21
22
University Health Network 23
6 Ming Feng Liu is currently located at University of California, San Francisco
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.01219-10 JVI Accepts, published online ahead of print on 22 September 2010
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Multi-Organ Transplant Program 24
585 University Avenue 25
Toronto, ON, M5G 2N2 26
Canada 27
28
*Ian D. McGilvray MD, PhD 29
Associate Professor of Surgery 30
Multi-Organ Transplant Program 31
University of Toronto 32
11C 1250 Toronto General Hospital 33
200 Elizabeth St, Toronto 34
Ontario, Canada 35
M5G 2C4 36
Phone: 416 340 5230 37
Fax: 416 340 5242 38
e-mail: [email protected] 39
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Abstract: 40
Ubiquitination is a critical regulator of the host immune response to viral infection, and 41
many viruses, including coronaviruses, encode proteins that target the ubiquitination 42
system. To explore the link between coronavirus infection and the ubiquitin system we 43
asked whether protein degradation by the 26S proteasome plays a role in severe 44
coronavirus infections using a murine model of SARS-like pneumonitis induced by 45
murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal 46
macrophages with inhibitors of the proteasome (PDTC, MG132, and PS-341) markedly 47
inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by 48
inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity 49
in macrophages, as well as the induction of inflammatory mediators such as IP-10, IFNγ 50
and MCP-1. In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in 51
A/J mice. Treatment of A/J mice with the proteasome inhibitors PDTC, MG132 or PS-52
341 led to 40% survival (p<0.01) with a concomitant improvement of lung histology, 53
reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and 54
reduced pulmonary inflammatory cytokine expression. These data demonstrate that 55
inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene 56
expression in vivo, by reducing MHV-1 replication and the resulting inflammatory 57
response. The results further suggest that targeting the proteasome may be an effective 58
new treatment for severe coronavirus infections. 59
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Introduction: 60
Severe acute respiratory syndrome (SARS), was first introduced into the human 61
population in the Guangdong Province in China and rapidly spread to several other 62
countries (31). SARS is caused by infection with the SARS coronavirus (SARS-CoV) 63
and is characterized by an atypical pneumonia and lymphopenia. Two-thirds of the 64
SARS-infected patients developed a viral pneumonitis, of which 10% developed acute 65
respiratory distress syndrome. During the outbreak in 2002 to 2003, 8,000 people were 66
infected and 774 people died from respiratory failure (36,44). At present there are no 67
effective treatments for SARS as well as other coronavirus infections. Finding an 68
effective treatment for coronavirus infections could protect us in the event of a re-69
emergent coronavirus outbreak (7). 70
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We have recently reported that a rodent model of SARS mimics many of the features 72
of severe clinical SARS pathology (11,12). Intranasal infection of A/J mice with strain 1 73
of murine hepatitis virus (MHV-1) causes a lethal form of pneumonitis characterized by 74
marked innate immune inflammatory cytokine production and replication of the virus in 75
pulmonary macrophages (11,12). MHV-1 infection is uniformly fatal in infected A/J 76
mice; the resultant disease serves as a model to understand the pathology of the most 77
severe SARS cases. In mice, the pulmonary damage is histologically similar to that seen 78
in human SARS, and is similarly associated with a marked upregulation of inflammatory 79
mediators, including MCP-1, IP-10, MIG, IFN-γ, IL-8 and IL-6 (11,12,25). These innate 80
immune mediators are likely to play roles in human SARS and MHV-1 SARS-like 81
pathogenesis. 82
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A critical aspect of the host innate immune response to viral illness is the 84
upregulation of the antiviral type 1 IFN response. With respect to SARS, type 1 IFN 85
responses have been reported to be suppressed by SARS-CoV in several models and in 86
clinical cases (11,39,46,53). In our model, MHV-1-infected A/J mice produce less type 1 87
IFN than resistant strains of mice, and they respond poorly to IFNβ therapy (11). Type I 88
IFN have been used clinically in the treatment of established SARS infections but have 89
shown only limited efficacy (25). In the absence of an effective antiviral treatment, the 90
innate immune pathways present a potential target for therapeutic intervention (7). 91
92
Ubiquitination, the process by which cellular proteins are conjugated to the 7.5Kb 93
ubiquitin protein, is a critical regulator of innate and adaptive immune pathways (40). 94
There are several possible fates for ubiquitinated proteins: degradation by the 26S 95
proteasome; trafficking to various subcellular sites; altered interactions with other 96
proteins; and, altered signal transduction functions (28). The fates of the ubiquitinated 97
proteins, many of which overlap, can play a role in innate immunity. From the first 98
discovery that papilloma virus encodes an E3 ubiquitin ligase that targets p53, it is now 99
widely appreciated that many viruses encode proteins that target or exploit ubiquitination 100
pathways (37,43). For example, Epstein Barr and herpes simplex proteins interact with 101
the host de-ubiquitinating protein, USP7 (13,17). Ubiquitination of IRF3 has been 102
implicated in the viral control of the innate immune system (22,49,50). Deubiquitination 103
(DUB) may also be important for viral functions, such as the assembly of viral replicase 104
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proteins with double-membrane vesicles at the site of replication, a process that 105
parasitizes autophagy (39). 106
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All coronaviruses, including MHV (A59 and JHM), infectious bronchitis virus, and 108
human CoV229E SARS coronavirus, encode one or more papain-like proteases, termed 109
PLpro (PL1pro and PL2pro) (3,5,19,23,51). One role for the PL2pro proteases is to 110
cleave the coronavirus polyprotein into its component parts. This enzyme isolated from 111
the SARS CoV has also been shown to have DUB activity both in vitro and in HeLa cells 112
(23) suggesting that it might also play a role in modulating the host ubiquitination 113
pathways. PLpro proteases harbor a N-terminal Ub-like domain reported to mediate 114
interactions between PLpro DUB activity and the cellular proteasome (35). Although 115
there is no direct link between the proteasome to SARS-CoV DUB activity, the presence 116
of the Ub1 domain and the SARS CoV DUB activity suggests that the proteasome may 117
be being exploited by the virus either to evade the immune response or to promote viral 118
replication. These interactions also suggest that the ubiquitination system might be a 119
target for anti-viral therapeutic intervention. 120
121
We explored the role of the cellular proteasome in MHV-1 replication and in the innate 122
immune response to the virus by testing the effects of small-molecule proteasome 123
inhibitors in both cell-based and murine models of SARS pneumonitis. We compared the 124
results in the SARS model to a well-described model of LCMV hepatitis in order to test 125
for virus-specific effects. To control for non-specific effects of the inhibitors, we used 126
three different agents: pyrollidine dithiocarbamate (PDTC), MG132 and PS-341 127
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(Bortezomib, VelcadeR). PDTC is a chelating agent that reversibly inhibits the 128
proteasome complex; MG132 is a peptide aldehyde protease inhibitor and PS-341 is a 129
peptide boronic acid inhibitor (1,20,38). PS-341 is a clinically approved drug, currently 130
being used in the treatment of multiple myeloma. 131
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Methods and Materials: 136
Animals, Buffers and Reagents. Pathogen-free female A/J mice aged 6-7 weeks were 137
purchased from Jackson, chow fed and allowed to acclimatize for at least 1 week prior to 138
experiments. 3% thioglycollate (Life Technologies, Inc.) was prepared as per the 139
manufacturer’s instructions. Endotoxin-free H21 and HBSS (Hanks Balanced Salt 140
Solution, Sigma) were obtained from Life Technologies, Inc. FBS was obtained from 141
HyClone. A 5mM pyrollidine dithiocarbamate (PDTC, Sigma) stock solution was 142
prepared in saline (pH 7.4); and a 20µM MG132 (Biomol) stock solution was prepared in 143
DMSO. PS-341 was synthesized by American Custom Chemicals Corp, San Diego CA 144
and prepared in DMSO at a concentration of 40mM. 145
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Cell and MHV-1 preparation. Peritoneal exudate macrophages (PEM) were harvested 147
in ice-cold HBSS 3 days following a 2ml intraperitoneal injection of 3% sterile 148
thioglycollate. Cells were washed twice in cold HBSS and resuspended in DMEM 149
(Gibco) 2%FCS, L-Gln at 1-10x106cells/ml. This procedure consistently yields a >96% 150
macrophage cell population by Wright’s stain, with >97% viability by trypan blue 151
exclusion (27). For most experiments 1x106 cells were plated on 6 well polystyrene plates 152
(Sarstedt) and allowed to incubate overnight at 37oC, 5%CO2. Non-adherent cells were 153
washed away with RPMI 1640 (Gibco), and replaced with RPMI/2%FCS/L-Gln. MHV-1 154
was obtained and purified as described previously (9). Virus was grown to titers of 10-50 155
x 106 PFU/ml H21 on confluent 17CL1 cells. 156
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Measuring PEM viability and MHV-1 viral replication. For studies of viral replication 158
in PEM, cells were pretreated for 60 minutes at 37oC, 5% CO2 in the presence or absence 159
of PDTC (50µM), MG132 (2µM) or PS-341 (0.1 µM). 1-18 hours after infection by 160
MHV-1 (multiplicity of infection, MOI=1), cells and culture media were harvested, 161
freeze-thawed at -20oC, and the virus titered on L2 cells as previously described (22). 162
Viability was measured by trypan blue exclusion on the Vi-CELL Series Cell Viability 163
Analyzer (Beckman Coulter, Mississauga, ON). 164
165
LCMV viral titers. 1×106 PEM were allowed to adhere to a 24-well plate for 4h. The 166
cells were then treated with either vehicle alone or a final concentration of 0.1 uM PS341, 167
2 uM MG-132 or 50 uM for PDTC for 60 min. After washing, the cells were treated with 168
LCMV-WE (gift from Pamela Ohashi, PMH) at an MOI of 1 for 1h followed by another 169
wash. At this point supernatant containing the proteasome inhibitor was added back to the 170
PEM. Cell culture supernatants were collected 18h post-infection and assayed for viral 171
titers using a plaque assay adapted from Battegay et al, (4). 172
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In vivo LCMV-WE infectious model. C57BL/6 mice were injected with LCMV-WE i.v. 174
at 2 ×106 pfu (n=5 per treatment group) or with vehicle alone (n=10). Animals were 175
treated immediately post-infection with vehicle or with one of the proteasome inhibitors 176
and every day following until sacrifice at day 8 p.i. (10µg MG132/dose, 100µg 177
PDTC/dose, or 5µg PS-341/dose and 0.5ml saline s.c.). Liver tissue samples were 178
collected and viral titers were assessed as described above (4). 179
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SARS pneumonitis model. As previously described (11), A/J mice were inoculated with 181
5000 PFU MHV-1 via an intranasal route. Mice were treated with PDTC (5mg/kg daily 182
s.c.), MG132 (0.5mg/kg daily s.c.), or PS-341 (0.25mg/kg daily s.c.) and 0.5ml saline s.c. 183
daily. All mice were monitored for signs of suffering and were euthanized at humane end 184
points according to protocols approved by the hospital animal care committee. Data was 185
analyzed using Graphpad Prism software (Graphpad Software). At various times after 186
infection plasma and serum were collected by cardiac puncture and lung tissue was 187
harvested and immersed in 10% formalin for H&E histology or prepared for real-time 188
PCR. 189
190
RNA isolation and Real Time PCR (qPCR). RNA was isolated using the TRIzol 191
method as per the manufacturer’s specifications (Invitrogen). RNA was reverse 192
transcribed using the First-Strand cDNA synthesis kit (Amersham Biosciences) using the 193
manufacturer’s protocol and the Amp 2400 PCR System (Perkin Elmer). qPCR was 194
performed with SYBR Green (Roche, Montreal, QC) on the LightCycler 480 system 195
(Roche) using a standard thermal cycling protocol. 384-well plates and optical covers 196
were purchased from Roche. Analysis was performed using LightCycler 480 software 197
using a Standard Curve relative quantification method. Samples were normalized to 198
HPRT (sense: 5'-agagggaaatcgtgcgtgac -3'; antisense: 5΄-gattcaacttgcgctcatcttaggc-3΄) 199
and β-actin (sense: 5'-actccaaagtttttatggctggt-3'; anti-sense: 5'-caatagtgatgacctggccgt) 200
housekeeping genes. PCR products were amplified with the following primer sequences: 201
IFNα (sense: 5'-actccaaagtttttatggctggt-3'; anti-sense: 5'-gtactgcccagaagtttcacatt); IFNγ 202
(sense: 5'-atgaacgctacacactgcatc-3'; antisense: 5'-ccatccttttgccagttcctc-3'); MCP-1 203
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(sense: 5'-ttaaaaacctggatcggaaccaa-3'; anti-sense: 5'-gcattagcttcagatttacgggt-3'); MIG 204
(sense: 5'-tccttttgggcatcatcttcc-3'; anti-sense: 5'-tttgtagtggatcgtgcctcg-3'); IFNβ (sense: 205
5'-tgaatggaaagatcaacctcaccta-3'; anti-sense: 5'ctcttctgcatcttctccgtca-3'); IP-10 (sense: 206
gccgtcattttctgcctcat-3'; anti-sense: 5'gcttccctatggccctcat-3't); TNFα (sense: 5'-207
catcttctcaaaattcgagtgacaa-3'; anti-sense: 5'tgggagtagacaaggtacaaccc-3'). 208
209
Western blot analysis. At various times after exposure to MHV-1 PEM were pelleted or 210
lung tissue was homogenized and lysed in ice-cold cell lysis buffer. Whole cell lysates 211
were prepared with 2x Laemmli, 0.1M dithiothreitol (DTT) buffer immediately followed 212
by incubation at 100oC for 5min. Cytosolic fractions were isolated with 1% TritonX-100, 213
150mM NaCl, 10mM Tris-HCl (pH 7.4), 2mM sodium orthovanadate, 10µg/ml 214
leupeptin, 50mM NaF, 5mM EDTA, 1mM EGTA, and 1mM phenylmethylsulfonyl 215
fluoride. Postnuclear supernatants were collected following centrifugation at 10,000x g 216
for 5min and diluted with 2x Laemmli buffer, 0.1mM DTT. Lysates prepared from 1×106 217
cells were separated on 12.5% SDS-PAGE and transferred to polyvinylidene difluoride 218
membrane (Millipore). Blots were probed with mouse monoclonal anti-MHV 219
nucleocapsid (kind gift of Dr. Julian Leibowitz, Texas A and M University, College 220
Station Texas); mouse monoclonal antibody to ubiquitinated proteins clone FK2 221
(Biomol); rabbit anti-mouse pStat1 (Tyr 701)-R (Santa Cruz); rabbit anti-mouse Stat1 222
p84/p91 (M-22) (Santa Cruz); and monoclonal anti-β-actin Clone AC-15 (Sigma). 223
Subsequently membranes were incubated with the corresponding horseradish peroxidase-224
conjugated secondary antibody: sheep-anti-mouse IgG – horseradish peroxidase linked 225
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(Amersham) or donkey anti-rabbit IgG-HRP (Santa Cruz Biotechnology). Blots were 226
developed using an ECL-based system (Amersham Pharmacia Biotech). 227
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Northern Blot Analysis. 6 hours post-infection with MHV-1, PEM were harvested and 229
RNA was isolated by the TRIzol method. MHV-1 RNA was separated on a 1.2% agarose 230
formaldehyde denatured gel and transferred onto Hybond-N+ (Amersham Biosciences) 231
nylon membrane. Northern blotting of RNA was performed according to the ExpressHyb 232
Manual (Clontech). The hybridization probes was amplified from the C-terminal of the 233
untranslated region of MHV-1 by RT-PCR using the following primers: sense: 234
MHV_UTR_B5' 5´– GAT GAA GTA GAT AAT GTA AGC GT –3´ and antisense: 235
MHV_UTR_3':5´– TGC CAC AAC CTT CTC TAT CTG TTA T –3´. DNA probes 236
(268bp) were labeled by random priming using Rediprime II Random Prime Labeling 237
System (Amersham Biosciences) according to manufacturer’s directions. Results were 238
analyzed using the GS-800 Calibrated Densitometer (Biorad) and Quantity One software 239
(Biorad). 240
241
Histology 242
Samples for histological analysis were fixed in 10% formalin and were processed
by 243
standard methods as described previously (11). Histological assessment for pulmonary 244
disease (pneumonitis, consolidation, peribronchial inflammation) was performed by a 245
pathologist (MJP) in a blinded, random manner. 246
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Results: 248
MHV-1 replication and cytotoxicity is blocked by inhibition of the cellular 249
proteasome. MHV-1 infects and replicates in A/J peritoneal PEM in culture, causing 250
cellular necrosis and the formation of large syncytia (data not shown). Necrotic cell death 251
plays a role in the tissue damage of severe coronavirus infections such as SARS (7,48). 252
Since coronaviruses express the PLP2 DUB enzyme which has been implicated in 253
coronavirus-induced pathogenesis (49), we tested the effect of inhibiting the function of 254
the cellular proteasome on viral cytotoxicity and replication of coronavirus. Therefore, 255
we pretreated PEM isolated from A/J mice with PDTC, MG132, or PS-341 for 1 hour 256
prior to infection with MHV-1. When PEM infected with MHV-1 were left untreated, the 257
level of polyubiquitination was decreased compared to the control PEM (uninfected and 258
untreated) and expressed high levels of viral nucleocapsid protein (N protein), an index of 259
MHV-1 replication (Figure 1A). However, in the presence of both MHV-1 infection and 260
proteasome inhibition, N protein expression was abrogated and cellular 261
polyubiquitination levels were similar to control groups treated with the inhibitor alone. 262
MHV-1 replication was also inhibited in treated cells when measured by viral titers 263
(Figure 1B). Decreased replication was associated with improved cellular viability 264
(Figure 1C) and improved cell morphology (data not shown). These data suggest that 265
proteasome inhibition negatively regulates viral replication and decreases the cytotoxic 266
effects of MHV-1 infection in PEM. 267
268
Proteasome inhibition has an effect on early MHV-1 replication. In order to 269
determine whether proteasome inhibition affects early or late stages of the MHV-1 life 270
cycle, we examined the time course of expression of viral RNA and protein in the 271
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presence and absence of proteasome inhibition. By Northern Blot analysis, infection of 272
PEM with MHV-1 in the presence of PDTC, MG132 or PS-341 decreased viral 273
replication as seen by the absence of subgenomic mRNA (Figure 2A). The marked 274
decrease in subgenomic viral RNA could be explained by an inhibition of viral entry into 275
the cell or an inhibition of viral replication. To test this hypothesis, we treated PEM with 276
PS-341 either 1 hour prior to infection or 1 hour post-infection with MHV-1 and 277
measured viral replication at several time points. The effect of proteasome inhibition was 278
observed 6h p.i. but not at earlier time points (data not shown). As measured by N protein 279
expression, viral replication was decreased at 6h post-infection whether cells were treated 280
with proteasome inhibitors before or after infection with MHV-1 indicating that viable 281
virus was present in both the proteasome inhibitor pre-treatment and the treatment p.i. 282
groups (Figure 2B). This finding suggests that MHV-1 entry or binding to PEM is not 283
dependent on the cellular proteasome. Together, these experiments demonstrate that 284
inhibition of the cellular proteasome targets early steps in coronavirus RNA replication 285
but not viral entry into the PEM. 286
287
Proteasome inhibition suppresses MHV-1 induced inflammatory cell activation. 288
Similar to SARS-mediated disease in humans, MHV-1 infection can induce a massive 289
and uncontrolled immune response in mice, initiated and driven by the induction of pro-290
inflammatory mediators (7,11). Pneumonitis, a characteristic symptom of MHV-1 291
induced disease, is driven by a pronounced innate immune response partly initiated and 292
amplified by pro-inflammatory cytokines (7). Therefore we tested whether proteasome 293
inhibition has an effect on virally-induced cellular activation as a potential mechanism of 294
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limiting disease pathogenesis. We measured the transcription levels of inflammatory 295
mediators found to be relevant to SARS and that are relevant to inflammatory responses: 296
IP-10, MCP-1, MIG-1, and TNFα (11). The mRNA levels for the four cytokines were 297
markedly increased following MHV-1 infection, but suppressed when proteasome 298
activity was inhibited (Figure 3A). The effect on cytokine expression might be due either 299
to decreased viral replication (Figure 1B), or to the acknowledged effect of proteasome 300
inhibitors on cytokine production (45), (10). To confirm that the proteasome inhibitors 301
can have a direct affect on cytokine expression in our system, we stimulated PEM with 302
bacterial endotoxin, 50ng/ml LPS, in the presence or absence of proteasome inhibition 303
(Figure 3B). Cytokine expression was measured by TNFα mRNA expression levels as 304
before. All proteasome inhibitors decreased TNFα expression following LPS stimulation. 305
Thus, the inhibition of the cellular proteasome affects MHV-1 replication, MHV-1 306
cytotoxicity, and inflammatory macrophage activation in vitro. 307
308
Proteasome inhibitor treatment improves survival of MHV-1 infected A/J mice. The 309
in vitro results mentioned in the previous sections suggest that inhibition of the cellular 310
proteasome has two potential benefits for the host: a decrease in viral replication, and 311
protection from virally induced inflammatory mediators. To explore whether the effects 312
of cellular proteasome inhibition might be translated to an in vivo system, we used a 313
murine SARS-like MHV-1 model and treated the infected mice with one of three 314
proteasome inhibitors: PDTC, MG132, and PS-341, the only proteasome inhibitor being 315
used clinically. The intranasal inoculation of A/J mice with 5000 PFU of MHV-1 has a 316
100% fatality rate (11). Using a treatment regimen of PDTC, MG132 or PS-341, the 317
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mortality rate of MHV-1 disease was reduced, with 40% of mice surviving long-term 318
(Figure 4A). 319
At day 7 p.i. with MHV-1, lung histology of untreated A/J mice showed severe peri-320
bronchitis and interstitial pneumonia affecting the entire lung which resulted in complete 321
lung consolidation followed by death (Table 1). PS-341 treated mice also developed peri-322
bronchitis and interstitial pneumonia, however at day 7, the percentage of the lung 323
involved (25-49%) decreased with a marked improvement in the area of the lung that was 324
consolidated (<25% in the PS-341 group compared to 100% consolidation in untreated 325
mice). Both PDTC and MG132 treated groups showed improved lung histology 326
compared to the untreated, MHV-1 infected control group. Overall, MG132 showed the 327
most prominent improvement in peri-bronchitis, interstitial pneumonia and lung 328
consolidation. These results suggest that SARS-like coronavirus infections are amenable 329
to treatment with agents that inhibit the proteasome in vivo. 330
331
Proteasome inhibition in vivo is associated with decreased production of pulmonary 332
inflammatory mediators. The increase in survival in the proteasome inhibitor treatment 333
groups observed in vivo might have been influenced by two key factors: decreased viral 334
replication decreased inflammatory cell activation, or a combination of both. Therefore, 335
we studied the effects of cellular proteasome inhibition on viral replication and on 336
activation markers of the innate immune response induced by MHV-1 in vivo. To this 337
end, mice were treated daily subcutaneously with a regimen of 5mg/kg daily for PDTC, 338
0.5mg/kg daily for MG132, or 0.25mg/kg for PS-341. Lung tissue was harvested at days 339
1.5, 4 and 7 days after inoculation of MHV-1 intranasally and tested for viral titers. All 340
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three proteasome inhibitors decreased pulmonary MHV-1 replication at all times studied 341
(data not shown); the most marked effect was seen following PS-341 treatment where 342
MHV-1 replication at day 7 post-infection decreased by 1.1 logs, p<0.01 (Figure 4B). In 343
addition, mRNA was extracted from the lung tissue to measure by Real Time PCR the 344
expression levels of IP-10, MCP-1, MIG, IFNγ, and TNF-α, as well as for Type 1 IFN. 345
PS-341 showed a consistent and marked inhibition of inflammatory mediator gene 346
expression, particularly IFNα (Figure 5). Taken together, these results suggest that a 347
modest effect on viral replication, coupled with a more marked effect on inflammatory 348
gene expression, contributes to the improved histology and outcomes seen in the in vivo 349
SARS model. This improvement was achieved despite a reduction in type 1 IFN gene 350
expression. 351
352
PS-341 delays expression of N-protein 353
In order to gain insight into the mechanisms underlying the inhibition of pulmonary 354
inflammatory mediator gene expression in vivo, we determined the pulmonary expression 355
of the N protein – one of the major mechanisms through which Coronaviruses influence 356
cellular activation (41,42,48). We also asked whether STAT1 phosphorylation was 357
altered in treated animals, since activation of the JAK/STAT cascade is upstream of the 358
induction of many inflammatory mediators and alterations in this signaling cascade have 359
previously been associated with inhibition of the cellular proteasome (26). As shown in 360
Figure 6, treatment of A/J mice with PS-341 significantly delayed the expression of N 361
protein in the lung and decreased the absolute amount of N protein. As well, STAT1 362
phosphorylation was delayed in treated mice. The ability of PS-341 to inhibit the mouse 363
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cellular proteasome in pulmonary tissue was confirmed by the increase in ubiquitinated 364
proteins at day 0.5 and 7. The in vivo innate immune response to MHV-1 in the presence 365
of PS-341 confirms our in vitro data that proteasome inhibition is an important factor 366
both in activating the innate immune response and facilitating viral replication. 367
368
Proteasome inhibition of viral replication is virus specific. 369
In order to test for virus-specific effects, we tested the efficacy of proteasome inhibition 370
on a second infectious agent, LCMV-WE, using a well-described model of LCMV 371
hepatitis in vivo and PEM infection in vitro (14,52). PEM infected with LCMV in vitro, 372
did not show a consistent decrease in replication when treated with PS-341, MG132 or 373
PDTC. PEM cultures that were treated with PS-341 did show slightly decreased MVH-1 374
production (Figure 7A). However, it is unlikely that this effect is a direct result of 375
proteasome inhibition per se since neither MG132 nor PDTC inhibited viral replication. 376
The general lack of effect of proteasome inhibition on LCMV replication in vitro was 377
mirrored in subsequent in vivo studies. C57BL/6 mice infected with LCMV-WE and 378
treated with proteasome inhibitors did not show a consistent decrease in viral titers 379
derived from liver tissue harvested at day 8 p.i. (Figure 7B). Interestingly, while PS-341 380
showed some degree of inhibition in vitro, the opposite effect was observed in vivo. 381
Given the consistent lack of effect of either PDTC and MG132, the effect of PS-341 on 382
LCMV replication is not likely to be due to proteasome inhibition per se, and contrasts 383
markedly with the inhibition of MHV-1 production (both in vitro and in vivo). 384
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Discussion: 386
In this study we present evidence that inhibition of the cellular proteasome has 387
important consequences for Coronavirus replication and innate immune activation. In 388
vitro, pretreatment of PEM with three proteasome inhibitors dramatically reduced viral 389
replication and the production of inflammatory mediators. In vivo the inhibition of the 390
proteasome had clear beneficial effects in the murine model of SARS, an effect that 391
seemed to be mediated by decreased inflammatory cell activation, as evidenced by a 392
reduction in inflammatory cytokine gene expression. Taken together these results suggest 393
that inhibition of the cellular proteasome could be considered as a therapy for SARS. 394
395
The fact that the SARS CoV papain-like protease (PLpro) has deubiquitinating 396
(DUB) activity both in in vitro studies and in HeLa cells emphasizes the link between 397
severe Coronavirus infections and ubiquitination-dependent pathways (3,23,49). PLpro 398
cleaves the coronavirus polyprotein at the N-terminal of the replicase gene at the 399
sequence LXGG, which is the consensus deubiquitination sequence targeted by other 400
DUB proteases such as USP14, HAUSP and UCH-L1. The SARS CoV and aIBV virus 401
encode only one PLpro, whereas all other Coronaviruses encode two PLpro (3), at least 402
one of which has the potential for DUB activity. The SARS CoV PLpro cleaves a 403
common ubiquitin substrate Ub-AMC, and deconjugates ISG15 (isopeptide bond 404
cleavage) both in cis and in trans (3,23). 405
406
Although Coronaviruses, including the SARS coronavirus, have the potential to 407
target ubiquitination pathways through a DUB protein, the role of the viral protease in 408
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deubiquitination remains unclear. Even though the SARS PLpro recognizes the LXGG 409
consensus de-ubiquitination sequence, the enzyme has much lower affinity for substrates 410
than other cellular DUB, and does not significantly contribute to cellular deubiquitination 411
(3,23). Inhibition of the SARS CoV DUB blocks coronavirus replication but less 412
effectively than the 26S proteasome inhibitors described here (34). 413
414
The mechanism through which proteasome inhibition disrupts MHV-1 replication 415
and activation of inflammatory cells is unclear, but is not likely due to an effect on viral 416
entry into the cell. PDTC, MG132 and PS-341 inhibited viral replication at the level of 417
RNA transcription. In vitro, the effect of proteasome inhibition was observed only after 6 418
hours of infection, and persisted even when introduced after infection of PEM (Figure 1). 419
In vivo, proteasome inhibition had relatively little effect on viral replication but did 420
attenuate inflammatory cytokine expression (Figure 5). Moreover, proteasome inhibition 421
also led to decreased redox activation but did not inhibit coronavirus-induced tyrosine 422
phosphorylation (data not shown), consistent with an effect focused more on the viral 423
replication machinery than on early viral signaling. Taken together these data suggest that 424
inhibition of the cellular proteasome leads to inhibition of MHV-1 replication and cellular 425
activation at steps after internalization of the virus. 426
427
Previous work has suggested that disrupting the cellular proteasome can also 428
inhibit the release of some strains of Coronaviruses into the cytoplasm from internalizing 429
lysosomes. Yu et al found that the release of the MHV-JHM strain into the cytoplasm 430
was sensitive to inhibition of the cellular proteasome with MG132 and lactacystin (47). In 431
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this study, treatment of cells with MG132 and lactacystin resulted in decreased MHV-432
JHM replication and accumulation of viral particles in late endosomes and lysosomes. 433
Although these effects may have been due to inhibition of the proteasome, there were no 434
detectable change in Ub-conjugated viral proteins or cellular proteins associated with 435
MHV, suggesting an alternative mechanism. In this regard, the authors noted that MG132 436
and lactacystin can also inhibit lysosomal proteins cathepsin B and A respectively 437
(2,20,24). 438
439
The beneficial effects of proteasome inhibition in the murine SARS model 440
correlate with an inhibition of cytokine production and improved histopathology more 441
than to a marked inhibition of viral replication. The cellular proteasome plays an 442
important role in macrophage inflammatory activation (18); indeed, in our model system 443
proteasome inhibition markedly decreases PEM cytokine production after exposure to 444
endotoxin (Supplementary Data). Based on these data one might expect some inhibition 445
of virally-induced macrophage activation, though this study is the first to our knowledge 446
to demonstrate this for coronaviruses. The consequences of this attenuation of 447
inflammatory cell activation will be mixed. Inhibiting aspects of the innate immune 448
response can ameliorate survival in models of coronavirus infection, even without an 449
effect on viral replication. For example, inhibition of the FGL2 membrane 450
prothrombinase, an important mediator of the innate immune response to MHV3-induced 451
fulminant hepatitis, improves survival without affecting early viral replication (21). The 452
interaction between viral replication, cytokine effects, and disease pathogenesis can be 453
complex: in the same model, inhibiting tyrosine kinase activation with Tyrphostin A59 454
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blocks some aspects of the innate immune response - hepatic expression of FGL2 - but 455
does not improve survival, possibly because viral replication is increased (ID McGilvray, 456
unpublished observations). Tissue damage resulting from coronavirus infection is the 457
result of both direct cell cytotoxicity and inflammatory-mediated destruction – both 458
mechanisms are important targets for therapy but either may, in certain contexts, play 459
more or less of a role. 460
461
In this study, the effect of the proteasome inhibitors in the murine model of SARS 462
is a global suppression of cytokine expression. This sort of suppression likely has both 463
positive and negative aspects. For example, many of the detrimental effects of SARS are 464
likely due to an overwhelming production of cytokines. In this case, suppression of 465
inflammatory cell activation would be expected to be beneficial. On the other hand, 466
pulmonary IFNα mRNA expression was decreased by all three proteasome inhibitors in 467
the murine model. Since IFNα is a key antiviral effectors, and one that is associated with 468
a positive clinical outcome (25), its suppression by the proteasome inhibitors may be one 469
reason that the effect of the proteasome inhibitors is not more marked. Other studies have 470
shown that the levels of type I IFN are suppressed following SARS-CoV infection both in 471
a proportion of SARS patients as well as several animal models (8,39,53). In the MHV-1 472
model of SARS, there is an induction of IFNα which is at first glance in contradiction to 473
these studies. However, as noted earlier, while there is induction of type 1 IFN in mice 474
susceptible to a SARS-like pneumonitis following MHV-1 infection, resistant mice 475
express more type 1 IFN – a pattern more in keeping with that suggested in studies of 476
SARS-CoV infection of peripheral blood monocytes and macrophages (46). In addition, 477
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cell and model-specific differences are likely to underlie some of the conflicting results. 478
We would expect that any intervention with the capacity to alleviate an otherwise 479
uniformly fatal model of SARS-like coronavirus infection would have that much more 480
effect in a less severe form of disease. 481
482
The effect of proteasome inhibition on viral infection and disease is likely to be 483
specific to the virus involved. For example, proteasome inhibition with PS-341 promotes 484
Epstein-Barr virus related gene expression in cell culture (6,15,16,54). We have found 485
that proteasome inhibition has little-to-no effect on replication of LCMV virus either in 486
vitro or in vivo (Figures 7A and 7B). Similarly, PS-341 has no effect on J6/JFH HCV 487
virus replication in Huh7.5 cells (ID McGilvray, unpublished data). Moreover, treatment 488
of clinical multiple myeloma with PS-341 may be associated with an increased rate of 489
vermicelli zoster virus reactivation (6). Interpreting the latter possibility is difficult, since 490
it may reflect the role of the cellular proteasome in either (or both) the viral infection and 491
the host response to the virus. Taken together, these examples illustrate the variability in 492
infectious routes and host responses and demonstrate that the role of the cellular 493
proteasome will vary with the specific virus in question. 494
495
Recently, two articles were published by the de Haan group, which assessed the 496
potential to use PS-341 as anti-CoV agent both in SARS-CoV infection and against MHV 497
infection in mice (32,33). Both the current study and those of the de Haan group 498
demonstrated an early decrease in viral replication with proteasome inhibition in vitro. 499
However, our study demonstrates a clear benefit to treatment of coronavirus infection in 500
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vivo using a MHV-1 pneumonitis model in A/J mice, while that of Raaben et al showed 501
increased viral titers and an adverse effect to PS-341 treatment in a MHV-A59 hepatitis 502
model in C57Bl/6 mice. There are a number of factors that differ between the studies and 503
likely contribute to the different outcomes. These factors include: the mouse strain (A/J in 504
our pneumonitis model, C57BL/6 in the Raaben et al hepatitis model); as well as the dose 505
(PS-341 0.25mg/kg/dose daily vs. 1mg/kg given day -1 and day 2 p.i.) and delivery route 506
(s.c. vs. i.p.). The viral strains differed as well: de Haan’s group used a recombinant 507
MHV-EFLM virus (derived from the A59 strain) or wild-type MHV-A59 leading to an 508
acute hepatitis in C57BL/6 while our study focused on a lung-tropic MHV-1 virus (with 509
no liver disease) in A/J mice. We have shown previously that different combinations of 510
coronavirus and host strains result in distinct outcomes in a variety of in vivo models 511
(11), 28, 29). For example, MHV-3, a coronavirus that causes an acute fulminant 512
hepatitis kills BALB/c mice within 3-4 days post-infection but is cleared by A/J mice and 513
has intermediate effects in C57BL/6 mice (29,30). Interestingly, with MHV-1 514
pneumonitis infection the opposite effect is seen: BALB/c and C57BL/6 mice are able to 515
clear the virus but, A/J mice succumb to the virus within 7-8 days post-infection (11). De 516
Albuquerque et al showed that MHV-1 induced disease in A/J resembles the pathology of 517
SARS and therefore serves as an ideal model for studying SARS-like disease. In this 518
model, our study demonstrates that PS-341 treatment increases survival, decreases viral 519
load and inhibits inflammatory cytokine expression. 520
521
In summary, this study presents evidence that MHV-1 replication and induction of 522
inflammatory cell activation can be attenuated by inhibition of the cellular proteasome. 523
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The inhibition of Coronavirus replication occurs at an early step, but not at the point of 524
viral entry into the cell. Proteasome inhibition has consequences both at the cellular and 525
whole animal level, with a similar inhibition of inflammatory cell activation in both 526
settings. The suppression of inflammatory cell activation seems to be particularly 527
important for the beneficial effect of proteasome inhibition in the murine SARS model. 528
Taken together these results suggest that proteasome inhibition is a novel therapeutic 529
intervention that could be considered in cases of clinical coronavirus infection. 530
531
Acknowledgments: 532
IDM is supported in part through a Canadian Institutes of Health Research operating 533
grant 62488. GL is supported by a Canadian Institutes of Health Research operating grant 534
CIHR 79561. AB has a scholarship from the University of Toronto Training Program in 535
Regenerative Medicine STP-53882. 536
537
538
539
540
541
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specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay? J 730
Exp. Med. 164:1075-1092. 731
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53. Zorzitto, J., C. L. Galligan, J. J. Ueng, and E. N. Fish. 2006. Characterization 732
of the antiviral effects of interferon-alpha against a SARS-like coronoavirus 733
infection in vitro. Cell Res 16:220-229. 734
54. Zou, P., J. Kawada, L. Pesnicak, and J. I. Cohen. 2007. Bortezomib induces 735
apoptosis of Epstein-Barr virus (EBV)-transformed B cells and prolongs survival 736
of mice inoculated with EBV-transformed B cells. J. Virol. 81:10029-10036. 737
738
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FIGURE LEGENDS 739
740
Figure 1 741
MHV-1 replication in PEM is inhibited by PS-341. PEM isolated from A/J mice were 742
infected with MHV-1 (MOI=1) and treated with three proteasome inhibitors: PS-341 (0.1 743
µM), MG132 (2µM) or PDTC (50µM). A) PS-341 treatment of MHV-1 infected PEM 744
inhibited N-protein expression and increased levels of ubiquitinated proteins by western 745
blot. Data is representative of two independent experiments. Each lane contains pooled 746
triplicate wells. B) PS-341 treatment decreased viral replication in MHV-1 infected PEM. 747
Data is represented as the log of the viral titer ± standard deviations (SD). C) PEM 748
viability was increased in the presence of PS-341, MG132 and PDTC. Data is represented 749
as % viability ± SD. Figures B and C represent are representative of three independent 750
experiments. Each sample was done in triplicate. 751
Figure 2 752
PS-341 inhibits transcription of viral proteins. 753
A) PS-341 inhibited the expression of MHV-1 subgenomic mRNA by Northern Blot. 754
PEM were harvested 6hr p.i. Each lane contains six pooled replicate wells. B) PS-341 755
effectively inhibits N protein expression when PEM were treated with the proteasome 756
inhibitor prior to- and post-infection with MHV-1. Each lane contains pooled triplicate 757
wells. Data is representative of two independent experiments. 758
Figure 3 759
PS-341 decreases levels of inflammatory cytokines in response to MHV-1 infection. 760
(A) mRNA expression levels of TNFα, IP-10, MCP-1 and MIG-1 in PEM decreased 761
following treatment with proteasome inhibitors as measured by Real time PCR. MCP-1 762
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and TNFα mRNA increased most prominently from the cytokines tested. Data is 763
representative of three independent experiments. Samples were run in triplicate. Data is 764
represented as fold increase ± SD. (B) Proteasome inhibitors downregulate TNFα mRNA 765
expression in PEM stimulated by LPS. PEM isolated from A/J mice were treated with 766
50ng/ml LPS and TNFα mRNA levels were measured by Real Time PCR after 6h. PEM 767
cultures were either pre-incubated for 1h with PDTC, MG132, PS-341 or left untreated. 768
TNFα mRNA expression was downregulated following treatment with any of the three 769
proteasome inhibitors. (n=3/group, data is representative of 2 independent experiments). 770
Figure 4 771
In vivo treatment of mice with PS-341 increases survival and attenuates viral 772
replication in vivo. A/J mice were infected with 5000 PFU MHV-1 intra-nasally and 773
then treated with PDTC (5mg/kg daily s.c.), MG132 (0.5mg/kg daily s.c.), or PS-341 774
(0.25mg/kg daily s.c.). (A) Survival of MHV-1 infected A/J increased significantly 775
following treatment with PS-341 (SEM was calculated using the Logrank test; *P=0.003, 776
**P=0.0003). n=10 for untreated and PS-341 treated animals; n=5 for MG132 and PDTC 777
treated animals. (B) Prolonged survival was associated with a 1.1 log decrease in viral 778
titers in PS-341-treated mice. Data represented as viral titers ± SD; P<0.01 between PS-779
341 treated and untreated mice (using a one-way ANOVA). n=10 for untreated group; 780
n=6 for PS-341 treated mice. 781
782
Figure 5 783
PS-341 treatment attenuates cytokine induction by MHV-1 in vivo. 784
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MHV-1 infection up-regulates the mRNA expression of IP-10, IFNγ, TNFα, MCP-1, 785
IFNβ and MIG in the lung. All the mentioned cytokines reach peak levels of expression 786
36h post-infection. In the presence of PDTC, MG132 and PS-341, cytokine mRNA levels 787
are markedly reduced. Particularly, PS-341 attenuates the mRNA expression levels more 788
dramatically then both PDTC and MG-132. n=3 for each group. Data is representative of 789
two independent experiments. 790
791
Figure 6 792
PS-341 alters expression and phosphorylation of viral and cellular proteins. Western 793
blot analysis of lung tissue lysate from MHV-1 infected mice. PS-341 treatment reduced 794
levels of viral protein (N protein) production compared to untreated controls. The protein 795
levels of STAT-1 remained unchanged between the PS-341 untreated and treated groups, 796
however the phosphorylation of STAT-1 was markedly decreased in PS-341 treated mice. 797
PS-341 treatment increased the levels of poly-ubiquitinated proteins. Data is 798
representative of three independent experiments. Each well contains pooled triplicate 799
wells. 800
Figure 7 801
LCMV-WE infection is unaffected by proteasome inhibition both in vitro and in 802
vivo. We tested the efficacy of proteasome inhibition on a second infectious agent, 803
LCMV-WE to assess the specificity of proteasome inhibition on coronavirus infections. 804
(A) 1×106 PEM were pre-treated with either vehicle alone or a final concentration of 0.1 805
uM PS341, 2 uM MG-132 and 50 uM for PDTC for 60min. The cells were washed and 806
subsequently infected with LCMV-WE at a MOI of 1. LCMV replication in vitro was not 807
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inhibited when treated with proteasome inhibitors. (B) C57BL/6 (n=5 per treatment 808
group, n=10 for vehicle alone) mice were infected i.v. with 2.0×106 pfu LCMV-WE. 809
Viral titers were assessed in livers collected from mice at day 8 post-infection when the 810
infection reaches peak levels (52). In contrast to MHV-1, LCMV-WE titers were not 811
inhibited by proteasome inhibition in vivo. 812
813
814
815
Table 1: Lung Pathology is improved following PS-341 treatment of MHV-1 mice 816
% of lung affected
Peri-bronchitis Interstitial Pneumonia Lung Consolidation
Days p.i. 1.5 4 7 1.5 4 7 1.5 4 7
Uninfected - - - - - - - - -
MHV-1 + ++ ++++ + ++ ++++ - + ++++
MHV-1+MG132 + + - ++ + + - - -
MHV-1+PDTC ++ - +++ +++ + +++ +++ - -
MHV-1+PS-341 +++ ++ ++ +++ ++ ++ +++ + +
(-) indicates no abnormal pathology, (+), (++), (+++) or (++++) indicate that <25%, 25-817
50%, 50-75% or 75-100% of the lung is affected respectively. 818
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Figure 4
A B
0 5 10 15 20 250
50
100MHV-1
MHV-1+PS341*
MHV1+MG132**
MHV1+PDTC**
Post-infection(day)
Perc
en
t S
urv
ival
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