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JOURNAL OF VIROLOGY Aug 2010 p 7880 7885 Vol 84 No 15 0022 538X 10 12 00 doi 10 1128 JVI 00486 10 Copyright 2010 American Society for Microbiology All Rights Reserved NOTES The Proteasome Inhibitor Velcade Enhances rather than Reduces Disease in Mouse Hepatitis Coronavirus Infected Mice H17188 Matthijs Raaben 1 Guy C M Grinwis 2 Peter J M Rottier 1 and Cornelis A M de Haan 1 Virology Division Department of Infectious Diseases and Immunology Faculty of Veterinary Medicine Utrecht University Utrecht Netherlands 1 and Pathology Division Department of Pathobiology Utrecht University Utrecht Netherlands 2 Received 4 March 2010 Accepted 7 May 2010 Many viruses including coronaviruses CoVs depend on a functional cellular proteasome for efficient infection in vitro Hence the proteasome inhibitor Velcade bortezomib a clinically approved anticancer drug shown in an accompanying study M Raaben et al J Virol 84 7869 7879 2010 to strongly inhibit mouse hepatitis CoV MHV infection in cultured cells seemed an attractive candidate for testing its antiviral properties in vivo Surprisingly however the drug did not reduce replication of the virus in mice Rather inhibition of the proteasome caused enhanced infection with lethal outcome calling for caution when using this type of drug during infection The cellular proteasome is a central actor in protein degra dation in eukaryotes 48 This barrel shaped cytosolic com plex has at least three peptidase activities i e caspaselike trypsinlike and chymotrypsinlike activities which are medi ated by different subunits 11 14 15 Proteasomal degrada tion is an important mechanism to control protein homeosta sis thereby regulating various basic cellular processes including cell cycle regulation cell adhesion gene transcrip tion and apoptosis 12 19 28 29 37 Because proteasome inhibitors induce apoptosis preferen tially in tumor cells they represent a novel class of drugs in anticancer therapy 10 16 30 42 One of these drugs Vel cade Bortezomib formerly known as PS 341 LDP 341 and MLM341 selectively blocks the chymotrypsinlike activity of the catalytic core of the proteasome 20S subunit 1 2 Vel cade has been clinically approved for the treatment of multiple myelomas and mantle cell lymphoma 17 18 while it also displays anticancer activity against a range of different human malignancies in cell culture and in animal models including myeloma pancreatic cancer lung cancer prostate cancer chronic lymphocytic leukemia and colon cancer 7 31 33 40 41 Viruses are obligatory intracellular parasites that exploit the host cell for generating their progeny virions A functional cellular proteasomal system has been shown to be critical for many viruses including coronaviruses CoVs at different stages of their life cycle 8 20 23 24 39 43 50 Thus in addition to their potential as anticancer drugs proteasome inhibitors also appear to be promising antiviral agents 39 45 Supporting this idea proteasome inhibitors have been shown to protect against coxsackievirus induced myocarditis and to prolong the survival of mice inoculated with Epstein Barr vi rus transformed B cells 13 51 However proteasome inhib itors have also been shown to exhibit immunosuppressive prop erties They can for example alter TLR4 induced dendritic cell activation and interfere with the immunological functions of T cells 4 27 Furthermore cancer patients treated with Velcade demonstrate increased reactivation of varicella herpes Corresponding author Mailing address Virology Division De partment of Infectious Diseases and Immunology Faculty of Veteri nary Medicine Utrecht University Yalelaan 1 3584 CL Utrecht Netherlands Phone 31 30 2534195 Fax 31 30 2536723 E mail c a m dehaan uu nl Present address Department of Microbiology and Molecular Ge netics Harvard Medical School 200 Longwood Avenue Boston MA 02115 5701 H17188 Published ahead of print on 19 May 2010 FIG 1 Velcade inhibits MHV infection in cell culture LR7 cells were pretreated with 10 H9262M Velcade or mock treated for 1 h Subse quently the cells were infected with MHV EFLM multiplicity of infection of 1 in the presence or absence of 10 H9262M Velcade After 1 h the inoculum was removed and the cells were extensively washed Incubation was then continued in the absence or presence of Velcade The virus titers in the culture media at the indicated time points were determined by using a quantal assay and are expressed as TCID 50 units 7880 on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from zoster virus infections 6 21 46 Thus while playing essential roles in the replication of several viruses the proteasome is also critically involved in generating an effective antiviral im mune response In the present study we evaluated the ability of Velcade obtained from Millennium Pharmaceuticals Inc to inhibit mouse hepatitis virus MHV infection in living animals Pre viously we showed that different proteasome inhibitors can dramatically affect the replication of different coronaviruses CoVs i e MHV FIPV and SARS CoV as determined by measuring reporter gene expression at different time points postinfection 33a We confirmed and extended these obser vations here by evaluating the antiviral effect of Velcade in cell culture by performing a one step growth curve with MHV A59 in LR7 mouse fibroblast cells 22 To this end cells were infected in the absence or presence of 10 H9262M Velcade Fig 1 At the indicated time points the virus titers in the supernatant were determined by using a quantal assay In the presence of Velcade virus production was dramatically affected Fig 1 a result that is in agreement with the decrease in virus replica tion observed previously 33a whereas cell viability was not affected as determined by a Wst 1 assay 47 To investigate whether the proteasome inhibitor Velcade could also inhibit infection in vivo we next used our recently developed bioluminescence imaging BLI model to monitor the spatial and temporal progression of MHV infection in living mice 35 To this end C57BL 6 mice were inoculated intraperitoneally with 10 6 50 tissue culture infective doses TCID 50 of MHV EFLM which is a recombinant MHV ex pressing the firefly luciferase FL reporter gene The mice were either pretreated with Velcade in phosphate buffered saline PBS at 1 mg kg or with an equal volume of PBS The drug was applied intraperitoneally on days H110021 and 2 relative to the time of inoculation with MHV EFLM Replication as measured by determining the amount of in vivo FL activity was assessed by BLI as described previously 35 All mice were imaged for exactly 10 min on their ventral sides As shown in Fig 2 treatment of MHV infected mice with Velcade resulted in a dramatic increase in FL expression This increase was already significant at day 2 postinfection but developed rapidly FIG 2 Bioluminescence imaging of MHV infected Velcade treated mice Six to eight week old C57BL 6 mice were injected intraperitoneally with Velcade 1 mg kg or PBS 30 min prior to inoculation with MHV EFLM Treatment with Velcade or PBS was repeated at day 2 Mice were anesthetized and subsequently imaged as described before using a Biospace CCCD camera 35 A Mice from each group were imaged simultaneously exactly 5 min after the injection of D luciferin Mice were imaged for 10 min on their ventral sides at days 2 and 4 postinfection p i B The bioluminescent signals expressed as counts were quantified for each group n H11005 8 using Photovision software Biospace Lab Note that two mice from the Velcade treated group succumbed to the infection before they could be imaged at day 4 while one mouse indicated by the asterisk died during imaging VOL 84 2010 NOTES 7881 on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from further during the next 2 days In sharp contrast the mock treated MHV infected animals showed a reduction in FL lev els from days 2 to 4 Furthermore the Velcade treated mice but not the mock treated animals showed severe clinical signs i e weight loss and rough fur with two mice already suc cumbing to the infection before day 4 data not shown These data strongly suggest that treatment of MHV infected mice with the proteasome inhibitor Velcade does not inhibit infec tion but rather enhances it To corroborate these results we subsequently performed an additional experiment in which we used more conventional methods to measure MHV replication in mice Therefore C57BL 6 mice were inoculated intraperitoneally with 10 6 TCID 50 of wild type MHV A59 Treatment with Velcade was performed as described above As an additional control a group of mice n H11005 4 was treated with Velcade only Body weight measurements were performed daily at day 4 postin fection all of the mice were sacrificed and the livers were collected to determine the viral loads The mice infected with MHV but not treated with Velcade and the uninfected Vel cade treated control animals did not show any clinical symp toms In contrast the animals infected with MHV and treated with Velcade showed severe clinical signs with a body weight drop at day 4 of ca 15 relative to their weight at the start of the experiment Fig 3A One mouse in this group already succumbed to the infection at day 3 The viral loads as mea sured by determining the PFU and the amounts of viral RNA in the liver homogenates Fig 3B and C were also higher in FIG 4 Velcade results in reduced infiltration of immune cells increased necrosis of hepatocytes and increased levels of MHV antigen A Isolated liver tissue was fixed in 4 neutral buffered formaldehyde and paraffin embedded Liver sections were routinely prepared for immunohistochemistry and analyzed for the presence of MHV antigen by staining with the polyclonal anti MHV serum k135 Representative images are shown for each experimental condition B and C Histopathological effects in the liver i e hepatocellular necrosis and the presence of inflammatory infiltrate were investigated by staining tissue sections with hematoxylin and eosin H E B or staining with an anti CD3 antibody C for identification of T lymphocytes Representative images are shown The dashed lines represent the borders of the lesions while the arrowheads indicate CD3 positive cells D The mRNA expression levels of several chemokines were determined in the liver homogenates by quantitative RT PCR using Assay On Demand reagents PE Applied Biosystems as described previously 36 The comparative C T method was used to determine the fold change for each gene The housekeeping gene encoding for 18S rRNA was used as a reference in all assays The levels of TNF H9251 CXCL 1 CXCL 2 IFN H9252 and IFN H9253 are expressed relative to their expression in livers from uninfected control animals Significant differences P H11021 0 05 in expression between mock and Velcade treated infected mice are indicated by the asterisk FIG 3 Velcade enhances MHV pathogenesis in mice Six to eight week old C57BL 6 mice were injected with Velcade 1 mg kg or PBS 30 min prior to infection with MHV A59 Control animals were not infected and were treated only with Velcade Treatment with Velcade or PBS was repeated at day 2 A Each of the following days the body weights of the animals were measured until the animals were euthanized at day 4 The body weights are expressed as percentages relative to the weights at the beginning of the experiment day 0 H11005 100 B The livers were isolated and homogenized as described previously 35 The amount of infectious virus in the homogenates was determined by plaque assays as described previously 35 The virus titers are expressed as PFU g of tissue the threshold of detection is 10 PFU g Note that the triangle indicated by the asterisk corresponds to a mouse that already succumbed to the infection during the night before day 4 C The relative amounts of viral genomic RNA in the liver homogenates were determined by quantitative TaqMan RT PCR on the 1b region of the MHV genome as described previously 9 7882 NOTES J VIROL on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from VOL 84 2010 NOTES 7883 on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from the animals treated with Velcade However the viral loads were not elevated as high as might have been expected on the basis of the BLI data Next we analyzed the expression of viral proteins in liver sections of the mice by staining with a polyclonal antiserum k135 raised against the structural proteins of MHV 38 Although we could detect only moderate staining of viral an tigen in the animals infected with MHV the lesions in MHV infected mice treated with Velcade appeared to be filled with viral protein Fig 4A In retrospect these data fit with the much higher expression of the FL reporter gene observed in the treated mice in the BLI experiment Fig 2 Histopatho logic evaluation of hematoxylin and eosin stained liver sec tions of the MHV infected mice showed randomly distributed coagulation necrosis of hepatocytes in mice treated with Vel cade and in the control group However light microscopic morphometric analysis using software from Olympus Soft Im aging Solutions Gmbh of the liver showed a significant P H11021 0 0001 3 fold increase in the surface area with necrosis in the MHV infected mice treated with Velcade compared to the control group 1 87 H11003 10 5 H9262m 2 H11006 4 1 H11003 10 4 H9262m 2 versus 5 89 H11003 10 4 H9262m 2 H11006 8 3 H11003 10 3 H9262m 2 respectively Moreover semiquan titative analysis of the inflammatory response showed a mark edly reduced presence of leukocytes in the necrotic areas of the liver in the MHV infected mice treated with Velcade com pared to the control animals Fig 4B Immunohistochemical evaluation of the affected areas indicated a reduction in T lymphocytes Fig 4C In agreement with previous results 34 we observed an upregulation of cytokine gene expression i e tumor necrosis factor alpha TNF H9251 CXCL 1 CXCL 2 and beta interferon IFN H9252 in the livers of MHV infected mice as determined by quantitative reverse transcription PCR RT PCR Interestingly a similar induction of cytokine gene ex pression was also observed in the MHV infected animals treated with Velcade with the exception of the IFN H9253 induc tion Fig 4D This latter observation probably reflects the absence of activated immune cells in the virus induced lesions Altogether we observed that proteasome inhibition during MHV infection results in an accumulation of viral antigen accompanied by the reduced presence of inflammatory cells at the sites of infection which results in a fatal coagulation ne crosis of hepatocytes The reduced inflammatory response most notably the re duced recruitment of T lymphocytes at the sites of MHV infection in the liver indicates that proteasome inhibition abrogates the induction of a protective immune response against MHV Likewise it has been shown recently that mice treated with Velcade were more susceptible to lymphocytic choriomeningitis virus infection 3 These observations corre spond with the findings of recent studies which demonstrate that proteasome inhibition promotes apoptosis in primary nat ural killer cells and dendritic cells DCs and inhibits DC maturation 26 44 49 In addition exposure of mice to bort ezomib radically impaired murine T lymphocyte development in the thymus 25 and induced selective apoptosis and de creased Th1 responses among alloreactive T lymphocytes while leaving unstimulated T cells unaffected 5 The present study shows for the first time that the immunosuppressive effects of the proteasome inhibitor Velcade may have a fatal outcome in mice infected with MHV indicating that protea some inhibitors are probably not useful as anti corona viral agents Our results warrant a more in depth investigation of the use of proteasome inhibitors in a clinical setting since they may result in adverse side effects in virus infected individuals This study was supported by grants from the M W Beijerinck Virology Fund Royal Netherlands Academy of Arts and Sciences and The Netherlands Organization for Scientific Research NWO VIDI 700 54 421 to C A M de Haan We thank Marne Hagemeijer and Mijke Vogels for stimulating discussions REFERENCES 1 Adams J 2002 Development of the proteasome inhibitor PS 341 Oncolo gist 7 9 16 2 Adams J and M Kauffman 2004 Development of the proteasome inhib itor Velcade Bortezomib Cancer Invest 22 304 311 3 Basler M C Lauer U Beck and M Groettrup 2009 The proteasome inhibitor bortezomib enhances the susceptibility to viral infection J Immu nol 183 6145 6150 4 Berges C H Haberstock D Fuchs M Miltz M Sadeghi G Opelz V Daniel and C Naujokat 2008 Proteasome inhibition suppresses essential immune functions of human CD4 H11001 T cells Immunology 124 234 246 5 Blanco B J A Perez Simon L I Sanchez Abarca X Carvajal Vergara J Mateos B Vidriales N Lopez Holgado P Maiso M Alberca E Villaron D Schenkein A Pandiella and J San Miguel 2006 Bortezomib induces selective depletion of 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NOTES J VIROL on March 7 2015 by GEO