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JOURNAL OF VIROLOGY Aug 2010 p 7869 7879 Vol 84 No 15 0022 538X 10 12 00 doi 10 1128 JVI 00485 10 Copyright 2010 American Society for Microbiology All Rights Reserved The Ubiquitin Proteasome System Plays an Important Role during Various Stages of the Coronavirus Infection Cycle H17188 Matthijs Raaben 1 Clara C Posthuma 2 Monique H Verheije 1 Eddie G te Lintelo 1 Marjolein Kikkert 2 Jan W Drijfhout 3 Eric J Snijder 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 Molecular Virology Laboratory Department of Medical Microbiology Leiden University Medical Center Leiden Netherlands 2 and Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden Netherlands 3 Received 4 March 2010 Accepted 7 May 2010 The ubiquitin proteasome system UPS is a key player in regulating the intracellular sorting and degra dation of proteins In this study we investigated the role of the UPS in different steps of the coronavirus CoV infection cycle Inhibition of the proteasome by different chemical compounds i e MG132 epoxomicin and Velcade appeared to not only impair entry but also RNA synthesis and subsequent protein expression of different CoVs i e mouse hepatitis virus MHV feline infectious peritonitis virus and severe acute respi ratory syndrome CoV MHV assembly and release were however not appreciably affected by these com pounds The inhibitory effect on CoV protein expression did not appear to result from a general inhibition of translation due to induction of a cellular stress response by the inhibitors Stress induced phosphorylation of eukaryotic translation initiation factor 2H9251 eIF2H9251 generally results in impaired initiation of protein synthesis but the sensitivity of MHV infection to proteasome inhibitors was unchanged in cells lacking a phosphorylat able eIF2H9251 MHV infection was affected not only by inhibition of the proteasome but also by interfering with protein ubiquitination Viral protein expression was reduced in cells expressing a temperature sensitive ubiquitin activating enzyme E1 at the restrictive temperature as well as in cells in which ubiquitin was depleted by using small interfering RNAs Under these conditions the susceptibility of the cells to virus infection was however not affected excluding an important role of ubiquitination in virus entry Our obser vations reveal an important role of the UPS in multiple steps of the CoV infection cycle and identify the UPS as a potential drug target to modulate the impact of CoV infection The cellular ubiquitin proteasome system UPS which is important for intracellular protein degradation in eukaryotic cells plays a central role in cellular protein homeostasis 59 64 Since all viruses exploit and manipulate the infrastructure and metabolism of their host cell to their own advantage it is not surprising that the UPS has also been implicated in the infection cycle and virus host interplay of several viruses 7 14 48 52 70 The UPS controls many different processes including the regulation of cell cycle progression apoptosis and antigen presentation 17 Proteins destined for proteasomal degrada tion are conjugated with chains of the small protein ubiquitin which constitute the recognition motif for the proteasome 21 In addition to targeting proteins for degradation conjugation with ubiquitin can also regulate intracellular protein sorting as has been described for numerous membrane proteins 22 Attachment of ubiquitin moieties to protein substrates occurs by the sequential action of three enzymes First the ubiquitin activating enzyme E1 forms a high energy thiolester bond with ubiquitin after which ubiquitin is transferred to the ubiquitin conjugating enzyme E2 Subsequently the ubiquitin is conju gated to a lysine side chain or to the N terminus of the sub strate by the corporate action of E2 and an E3 ubiquitin ligase with the latter enzyme determining the substrate specificity of the process Subsequently the UPS targets these polyubiq uitinated substrates to the catalytic 20S core complex of the proteasome which subsequently cleaves them into smaller peptides The proteasome controls not only hydrolysis of functionally active proteins but also the degradation of mis folded polypeptides Coronaviruses CoVs are enveloped positive strand RNA viruses and are common pathogens in many animal species With a size of 28 to 32 kb CoVs have the largest genome among RNA viruses known to date Several CoVs cause severe disease in animals including porcine transmissible gastroen teritis virus bovine coronavirus avian infectious bronchitis viruses and feline infectious peritonitis virus FIPV With the discovery of new human CoVs HCoVs such as the severe acute respiratory syndrome SARS CoV 15 HCoV NL63 62 and HCoV HKU1 67 interest in CoV research has significantly increased The well studied mouse hepatitis virus MHV is often used as a model CoV The CoV infection cycle starts with the attachment of the virus to a specific cellular receptor The spike S protein a class I fusion protein is responsible for virus entry by mediat ing both receptor binding and the subsequent fusion of the viral envelope with a host membrane 6 10 After virus entry 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 Division Pathology Department Pathobiology Utrecht University Yalelaan 1 3584 CL Utrecht Netherlands H17188 Published ahead of print on 19 May 2010 7869 on March 7 2015 by ST ANDREWS UNIV http jvi asm org Downloaded from the viral genome is released into the cytosol of the cell where it is translated into two large replicase polyproteins These are autoproteolytically processed to produce 15 or 16 mature non structural proteins nsp s which assemble into viral replica tion transcription complexes that are thought to be associated with a virus induced network of modified endoplasmic re ticulum ER membranes which includes double membrane vesicles and other unusual membrane structures 18 31 54 63 Subsequently a nested set of sub genomic mRNAs is produced 42 which are translated into the viral structural and accessory proteins Together with the newly synthesized genomic RNA the structural proteins assemble into progeny virions by budding through membranes of the ER to Golgi intermediate compartment ERGIC 32 The newly synthe sized virions are subsequently released by exocytosis In the present study we investigated the importance of the UPS during CoV infection Besides a previous study reporting that inhibition of the proteasome affected MHV entry 68 no comprehensive analysis of the involvement of the UPS in the CoV replicative cycle has been performed until now Here we interfered with the UPS either by treating cells with chemical inhibitors of the proteasome by using cells that express a temperature sensitive form of the ubiquitin activating enzyme E1 or by knockdown of ubiquitin synthesis with small inter fering RNAs siRNAs Whereas CoV RNA synthesis and subsequent protein expression were severely reduced under all experimental conditions tested virus entry appeared only to be affected by the chemical inhibitors of the proteasome MATERIALS AND METHODS Cells and viruses Murine LR7 34 feline FCWF and Vero E6 cells were used to propagate the viruses i e recombinant MHV FIPV and SARS CoV respectively and for infection experiments All work with live SARS CoV was performed inside biosafety cabinets in the biosafety level 3 facility at Leiden University Medical Center Chinese Hamster E36 and ts20 cells 33 were main tained at 31 C in H9251 minimal essential medium supplemented with 10 vol vol fetal calf serum Bodinco B V 100 U of penicillin ml and 100 H9262g of strepto mycin ml The mouse embryonic fibroblasts MEFs expressing wild type or mutant S51A eIF2H9251 50 and HeLa CEACAM1a 63 cells were maintained in complete Dulbecco modified Eagle medium Cambrex BioScience containing 10 vol vol fetal calf serum Bodinco B V 100 U of penicillin ml and 100 H9262g of streptomycin ml supplemented with 1H11003 nonessential amino acids Invitro gen MHV EFLM 13 MHV nsp2EGFP 63 FIPV H90043abcFL 13 and SARS CoV GFP 53 were used for the infection experiments Chemicals Stocks of 10 mM MG132 1 mM epoxomicin and 5 mM lactacystin all obtained from Sigma Aldrich were prepared in dimethyl sulfoxide DMSO A stock of 1 mM Velcade Millennium Pharmaceuticals Inc was prepared in phosphate buffered saline PBS All stocks were stored at H1100220 C Luciferase assays Cell monolayers infected with FL expressing viruses were lysed at the indicated times postinfection using the appropriate buffer provided with the firefly luciferase FL assay system Promega Intracellular luciferase expression was measured according to the manufacturer s instructions and the relative light units RLU were determined with a Berthold Centro LB 960 plate luminometer Confocal immunofluorescence microscopy Cells were fixed with 4 parafor maldehyde in PBS and subsequently permeabilized with 0 1 Triton X 100 in PBS When indicated the MHV infected cells were incubated for 1 h with the first antibody directed against nsp2 3 kindly provided by Susan Baker 25 or against double stranded RNA dsRNA English and Scientific Consulting Bt K1 51 diluted in PBS containing 10 normal goat serum After several washing steps the cells were incubated with an appropriate dilution of secondary antibody in the same buffer for 1 h After three subsequent washing steps the coverslips were mounted in FluorSave Calbiochem The immunofluorescence staining was analyzed by using a confocal laser scanning microscope Leica Green fluorescent protein GFP was excited at 488 nm Cy3 at 568 nm and Cy5 at 633 nm Metabolic labeling and immunoprecipitation At 4 5 h postinfection the MHV infected cells were starved for 30 min in cysteine and methionine free modified Eagle medium containing 10 mM HEPES pH 7 2 and 5 dialyzed fetal calf serum The medium was then replaced by the same medium containing 100 H9262Ci of 35 S in vitro cell labeling mixture Amersham Biosciences after which the cells were further incubated for 30 min Subsequently the cells were either lysed or incubation was continued with culture medium chase The cells were lysed and cell lysates were subjected to immunoprecipitation as described pre viously 40 using polyclonal antisera directed against MHV k135 and the M protein anti M c 35 Culture supernatants were subjected to immunoprecipi tation in the absence of detergents using the A3 10 monoclonal antibody directed against the S protein 66 The immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE and autora diography Reporter RNA synthesis and transfection The reporter plasmid pM5f RL M3 63 was linearized by using a PacI restriction site directly downstream of the poly A sequence Subsequently RNA transcripts were produced by using the T7 MessageMachine kit Ambion according to the manufacturer s instructions Next 0 5 pmol of RNA was transfected into cells by using Lipofectamine 2000 Invitrogen Cells were treated with 10 H9262g of MG132 ml or mock treated for 4 h after which the cells were lysed and Renilla luciferase activity was measured with a Renilla luciferase assay kit Promega according to the manufacturer s protocol Immunocytochemistry Cell monolayers were fixed permeabilized and pro cessed for immunocytochemistry as described previously 12 Peroxidase was visualized by using an AEC substrate kit from Vector Laboratories MHV positive cells were detected and counted by using bright field light microscopy SARS CoV nsp5 protease assay To obtain recombinant SARS CoV main protease M pro nsp5 was expressed in Escherichia coli BL21 DE3 from expres sion vector pMal SARS CoV M pro His as the C terminal domain of a maltose binding protein MBP fusion protein 24 Autocatalytic cleavage of the fusion protein during expression liberated the authentic nsp5 N terminus At its C terminus recombinant nsp5 was extended with a His 6 tag to allow for affinity chromatography purification of the protein As a negative control for enzymatic activity a C145A mutant nsp5 1 was expressed from the same plasmid wild type and mutant plasmids were kindly provided by John Ziebuhr and Tanja Schirmeister Expression of SARS CoV wild type nsp5 and the C145A mutant was induced by using autoinduction medium ZYM 5052 57 and recombinant proteins were purified by using Talon metal affinity resin beads Clontech as previously described 24 Fluorimetric in vitro activity assays were performed at 30 C using a synthetic 8 amino acid peptide substrate labeled with a fluorescence resonance energy transfer pair 24 and containing a consensus CoV M pro cleav age site Val X Leu Asn2Ser In a 100 H9262l reaction volume 0 43 H9262g of purified nsp5 and 50 H9262M the Dabcyl VRLQSGTC fluorescein peptide substrate were incubated in a 20 mM Tris HCl buffer pH 7 5 containing 0 1 mM EDTA 1 mM dithiothreitol 200 mM NaCl and 12 5 DMSO The increase in fluorescence 485 nm excitation 535 nm emission resulting from cleavage of the substrate was measured for 60 min in a Mithras LB940 fluorimeter Berthold in the presence or absence of Velcade Cleavage rates were calculated by fitting a linear curve through data obtained during the first 10 min of the assay The cleavage rates are expressed in dF min i e the change in fluorescence per minute Ubiquitin knockdown siRNA duplexes targeting different sites within the coding sequences of UBA52 and RPS27A were designed by and obtained from Ambion Inc three siRNAs per gene Scrambled siRNAs or siRNAs targeting FL GL2H11001GL3 all from Ambion were taken along as controls in each exper iment One day after seeding the HeLa CEACAM1a cells were transfected with a final concentration of 10 nM siRNA using Oligofectamine Invitrogen At 72 h after transfection the cells were inoculated with MHV EFLM At 6 h postin fection the cell number and viability was measured by Wst 1 assay according to the manufacturer s protocol Roche Diagnostics GmbH Subsequently intra cellular luciferase expression was determined as described above Each siRNA experiment was performed in triplicate For each well RLU values were cor rected for cell number and viability as determined by the Wst 1 assay Western blotting Depletion of ubiquitin after siRNA transfection was con firmed by Western blotting To this end cells were lysed in ice cold lysis buffer 20 mM morpholinepropanesulfonic acid pH 7 2 5 mM EDTA 2 mM EGTA and 0 5 wt vol Nonidet P 40 containing 30 mM NaF 40 mM H9252 glycerophos phate 20 mM sodium pyrophosphate 1 mM sodium orthovanadate 1 mM phenylmethylsulfonyl fluoride 3 mM benzamidine 1 5 H9262M pepstatin A and 10 H9262M leupeptin Cell lysates were cleared by centrifugation at 100 000 H11003 g at 4 C for 30 min Proteins present in the cell lysates were separated by SDS PAGE and transferred to a nitrocellulose membrane 0 1 H9262M Schleicher Santa Cruz Biotechnology Inc After exten sive washing of the membrane the amount of protein was visualized and quan titated by using an Enhanced ChemoLuminescence Plus kit a Typhoon imager and ImageQuant TL software all from Amersham Biosciences Immunoblot ting for endogenous levels of eIF2H9251 and eIF2H9251P was performed as described previously 43 RESULTS MG132 inhibits MHV at multiple steps of the infection cycle We started our analysis of the role of the UPS in the CoV replicative cycle by studying the one step growth of MHV strain A59 MHV A59 in LR7 cells in the absence or presence of the well known proteasome inhibitor MG132 In this assay the effect of inhibition of proteasome activity on different steps of the infection cycle was investigated To this end LR7 cells were inoculated with MHV EFLM in the presence or absence of 10 H9262M MG132 MHV EFLM is a recombinant virus that expresses the FL reporter gene from a subgenomic mRNA 13 The intracellular FL level which is an indirect measure for viral RNA synthesis 43 63 was significantly delayed in the presence of MG132 at 6 9 and 12 h postinfection Fig 1A As a likely consequence the extracellular accumulation of progeny virions at these time points was also dramatically af fected Fig 1B To investigate which specific step s of the MHV infection cycle might be affected by the lack of proteasome activity we evaluated the effects of adding MG132 to the culture medium at different time points First we determined the dose re sponse curves when the drug was applied either already during i e from 0 to 6 h postinfection or after the virus inoculation period i e from 2 to6horfrom 2 to 8 h postinfection As shown in Fig 1C virus replication i e the ability to reproduce in cells was much more affected when the drug was already present during virus inoculation 50 inhibitory concentration IC 50 H11005 0 17 H9262M than upon addition of the drug at 2 h postinfection IC 50 H11011 0 5 H9262M The duration of the MG132 postincubation period did not appear to affect the IC 50 com pare the periods from 2 to 6 and from 2 to 8 h These results suggest that early steps in the virus life cycle as well as later steps which include viral RNA synthesis and subsequent pro tein expression are affected by the inhibition of proteasome activity Confocal microscopy was next used to confirm the inhibition of MHV RNA synthesis and subsequent protein expression by MG132 To this end an infection experiment was performed with a recombinant virus that expresses nsp2 fused to en hanced GFP EGFP from a subgenomic mRNA 19 This fusion protein is recruited to the virus induced membrane net work with which viral RNA synthesis is thought to be associ ated In this experiment LR7 cells infected with MHV nsp2EGFP were mock treated or treated with MG132 from 2 to 7 h postinfection Subsequently the cells were fixed and stained for two additional markers of the putative replication sites by using antibodies directed either against nsp2 and nsp3 or against dsRNA In agreement with our previous results in the presence of MG132 the staining for nsp2 3 dsRNA and nsp2EGFP was dramatically reduced in comparison with the untreated control cells Fig 1D Interestingly it appeared that the expression of nsp2EGFP was more affected than that of nsp2 3 by treatment with MG132 since barely any EGFP fluorescence could be detected Although nsp2 3 is expressed directly from the viral genome the nsp2EGFP fusion protein is expressed from a subgenomic viral mRNA and hence tran scription is required for its expression These results therefore strengthen the conclusion that MG132 affects MHV RNA syn thesis The UPS has been demonstrated to play an important role in the assembly and release of some viruses 20 56 60 65 The reduction of extracellular infectivity shown in Fig 1B may indicate a similar effect of MG132 on assembly of MHV Therefore we studied the effect of the drug on the synthesis and release of MHV particles by using an assembly assay In this assay cells infected with MHV were metabolically labeled from 5 to 5 5 h postinfection in the absence of MG132 Sub sequently the cells were chased in the presence of cyclohexi mide to inhibit protein synthesis and in addition they were mock treated or treated with MG132 After 30 min i e at 6 h postinfection the chase medium was refreshed and the re lease of newly assembled viral particles was monitored from 6 to 8 h and from 8 to 10 h postinfection Virus particles were affinity isolated from the culture media harvested at 8 or 10 h postinfection using antibodies to the S protein At 10 h postin fection cells were lysed and cell lysates were monitored for viral protein production As shown in Fig 1E no appreciable effect of MG132 on virus assembly could be observed Only in th