1 1 2 3 Proteasome inhibition in vivo promotes survival in a lethal ...

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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

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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

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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

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*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

146

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

173

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

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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

228

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

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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

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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

542

543

544

545

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52. Zinkernagel, R. M., E. Haenseler, T. Leist, A. Cerny, H. Hengartner, and A. 727

Althage. 1986. T cell-mediated hepatitis in mice infected with lymphocytic 728

choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-729

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 1

A

B C


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B


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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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