key: cord-336066-n9yq8enz
authors: Lai, Chien‐Chen; Jou, Ming‐Jia; Huang, Shiuan‐Yi; Li, Shih‐Wein; Wan, Lei; Tsai, Fuu‐Jen; Lin, Cheng‐Wen
title: Proteomic analysis of up‐regulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus 3C‐like protease
date: 2007-04-04
journal: Proteomics
DOI: 10.1002/pmic.200600459
sha: 
doc_id: 336066
cord_uid: n9yq8enz

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS CoV) is an important issue for treatment and prevention of SARS. Previously, SARS CoV 3C‐like protease (3CLpro) has been demonstrated to induce apoptosis via the activation of caspase‐3 and caspase‐9 (Lin, C. W., Lin, K. H., Hsieh, T. H., Shiu, S. Y. et al., FEMS Immunol. Med. Microbiol. 2006, 46, 375–380). In this study, proteome analysis of the human promonocyte HL‐CZ cells expressing SARS CoV 3CLpro was performed using 2‐DE and nanoscale capillary LC/ESI quadrupole‐TOF MS. Functional classification of identified up‐regulated proteins indicated that protein metabolism and modification, particularly in the ubiquitin proteasome pathway, was the main biological process occurring in SARS CoV 3CLpro‐expressing cells. Thirty‐six percent of identified up‐regulated proteins were located in the mitochondria, including apoptosis‐inducing factor, ATP synthase beta chain and cytochrome c oxidase. Interestingly, heat shock cognate 71‐kDa protein (HSP70), which antagonizes apoptosis‐inducing factor was shown to down‐regulate and had a 5.29‐fold decrease. In addition, confocal image analysis has shown release of mitochondrial apoptogenic apoptosis‐inducing factor and cytochrome c into the cytosol. Our results revealed that SARS CoV 3CLpro could be considered to induce mitochondrial‐mediated apoptosis. The study provides system‐level insights into the interaction of SARS CoV 3CLpro with host cells, which will be helpful in elucidating the molecular basis of SARS CoV pathogenesis.

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS CoV) is an important issue for treatment and prevention of SARS. Previously, SARS CoV 3C-like protease (3CLpro) has been demonstrated to induce apoptosis via the activation of caspase-3 and caspase-9 (Lin, C. W., Lin, K. H., Hsieh, T. H., Shiu, S. Y. et al., FEMS Immunol. Med. Microbiol. 2006, 46, 375-380) . In this study, proteome analysis of the human promonocyte HL-CZ cells expressing SARS CoV 3CLpro was performed using 2-DE and nanoscale capillary LC/ESI quadrupole-TOF MS. Functional classification of identified up-regulated proteins indicated that protein metabolism and modification, particularly in the ubiquitin proteasome pathway, was the main biological process occurring in SARS CoV 3CLpro-expressing cells. Thirty-six percent of identified up-regulated proteins were located in the mitochondria, including apoptosis-inducing factor, ATP synthase beta chain and cytochrome c oxidase. Interestingly, heat shock cognate 71-kDa protein (HSP70), which antagonizes apoptosis-inducing factor was shown to down-regulate and had a 5.29-fold decrease. In addition, confocal image analysis has shown release of mitochondrial apoptogenic apoptosis-inducing factor and cytochrome c into the cytosol. Our results revealed that SARS CoV 3CLpro could be considered to induce mitochondrial-mediated apoptosis. The study provides system-level insights into the interaction of SARS CoV 3CLpro with host cells, which will be helpful in elucidating the molecular basis of SARS CoV pathogenesis.

A novel virus, severe acute respiratory syndrome (SARS)associated coronavirus (SARS CoV) is rapidly transmitted through aerosols, causing 8447 reported SARS cases with 811 deaths worldwide in a short period from February to June, 2003 [1] [2] [3] [4] [5] . The SARS patients had manifested symptoms, like bronchial epithelial denudation, loss of cilia, multinucleated syncytial cells and squamous metaplasia in their lung tissue [6, 7] . Other studies have shown that SARS CoV replicates in Vero-E6 cells with cytopathic effects [8, 9] , and induces AKT signaling-mediated cell apoptosis [10] .

SARS CoV particles contain an approximately 30-kbp positive-stranded RNA genome with a 5' cap structure and a 3' poly(A) tract [11] [12] [13] . The SARS CoV genome encodes replicase, spike, envelope, membrane, and nucleocapsid proteins. The replicase gene encodes two large overlapping polypeptides (replicase 1a and 1ab, ,450 and ,750 kDa, respectively), including 3C-like protease (3CLpro), RNA-dependent RNA polymerase, and RNA helicase for viral replication and transcription [14] . The SARS CoV 3CLpro mediates the proteolytic processing of replicase 1a and 1ab into functional proteins, playing an important role in viral replication. Therefore, the SARS CoV 3CLpro is an attractive target for developing effective drugs against SARS [12] [13] [14] . Recently, a SARS CoV 3CLpro-interacting cellular protein, vacuolar-H1 ATPase (V-ATPase) G1 subunit with a 3CLpro cleavage site-like motif was identified, affecting the intracellular pH in 3CLpro-expressing cells [15] . In human promonocyte cells, SARS CoV 3CLpro has been demonstrated to induce apoptosis via caspase-3 and caspase-9 activities [16] . In addition, 3C protease of picornaviruses poliovirus, enterovirus 71 and rhinovirus have been demonstrated to be associated with host translation shutoff by cleaving the translation initiation factor eIF4GI and the poly(A)-binding protein (PABP) [17] , and inactivation of NF-kappaB function by proteolytic cleavage of p65-RelA [18] . Apparently, SARS CoV 3CLpro plays a pivotal role in the pathogenesis processes. Therefore, investigating pathogenesis of SARS CoV 3CLpro has become an important issue.

In the post-genomic era, the combination of 2-DE and MS has provided an alternative approach to examine a comparative analysis of proteomic profiling during viral infection, allowing new insights into cellular mechanisms involved in viral pathogenesis [19] [20] [21] [22] [23] [24] . The 2-DE/MS proteomic technologies have been used to analyze the protein profiles of plasma from SARS patients [19, 24] , and to differentiate up-regulated and down-regulated proteins in SARS CoV-infected African green monkey kidney cells [20] . To identify proteomic alternations induced by SARS CoV 3CLpro, the combination of 2-DE and MS can be performed for quantitative analysis and identification of the unique protein profiling in the transfected cells-expressing 3CLpro.

In this study, we intended to investigate the comparative proteome analysis of human promonocyte HL-CZ cells in the presence and absence of SARS CoV 3CLpro. Seventythree up-regulated and 21 down-regulated proteins identified in the 3CLpro-expressing cells were categorized according to their subcellular location, biological process and biological pathway based upon the PANTHER classification system (http://www.pantherdb.org/). Functional analysis of up-regulated proteins identified in the 3CLpro-expressing cells was further examined using immunoblot analysis and confocal microscopy.

In our previous study [16] , human promonocyte HL-CZ cell clone co-transfected with the plasmid p3CLpro plus indicator vector pEGFP-N1 was established for 3CLpro-expressing cells, whereas human promonocyte HL-CZ cell clones cotransfected with the plasmid pcDNA3.1 plus indicator vector pEGFP-N1 were used as mock cells. The transfected cells were incubated with RPMI 1640 medium containing 10% FBS and 800 mg/mL of antibiotic G418. For determining expression of SARS CoV 3CLpro, the transfected cells were analyzed using Western blotting. The cell lysates were dissolved in 2X SDS-PAGE sample buffer without 2-mercaptoethanol, and boiled for 10 min. Proteins were resolved on 12% SDS-PAGE gels and transferred to NC paper. The resultant blots were blocked with 5% skim milk, and then reacted with appropriately diluted mouse mAb anti-His tag (Serotec), anti-Rpt4 (26S protease regulatory subunit 6A) (abcam) or rabbit anti-apoptosis-inducing factor (Sigma) for a 3-h incubation. The blots were then washed with TBST three times and overlaid with a 1/5000 dilution of alkaline phosphatase-conjugated with secondary antibodies. Following 1-h incubation at room temperature, blots were developed with TNBT/BCIP (Gibco).

For 2-DE, mock cells and 3CLpro-expressing cells were harvested, washed twice with ice-cold PBS, and then extracted with lysis buffer containing 8 M urea, 4% CHAPS, 2% pH 3-10 non-linear (NL) IPG buffer (GE Healthcare), and the Complete, Mini, EDTA-free protease inhibitor mixture (Roche). After a 3-h incubation at 47C, the cell lysates were centrifuged for 15 min at 16 0006g. The protein concentration of the resulting supernatants was measured using the BioRad Protein Assay (BioRad, Hercules, CA, USA). Protein sample (100 mg) was diluted with 350 mL of rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer pH 3-10 NL, 18 mM DTT, 0.002% bromophenol blue), and then applied to the nonlinear Immobiline DryStrips (17 cm, pH 3-10; GE Healthcare). After the run of 1-D IEF on a Multiphor II system (GE Healthcare), the gel strips were incubated for 30 min in the equilibration solution I (6 M urea, 2% SDS, 30% glycerol, 1% DTT, 0.002% bromophenol blue, 50 mM Tris-HCl, pH 8.8), and for another 30 min in the equilibration solution II (6 M urea, 2% SDS, 30% glycerol, 2.5% iodoacetamide, 0.002% bromophenol blue, 50 mM Tris-HCl, pH 8.8). Subsequently, the IPG gels were transferred to the top of 12% polyacrylamide gels (20620 cm61.0 mm) for the secondary dimensional run at 15 mA, 300 V for 14 h. Separated protein spots were fixed in the fixing solution (40% ethanol and 10% glacial acetic acid) for 30 min, stained on the gel with silver nitrate solution for 20 min, and then scanned by GS-800 imaging densitometer with PDQuest software version 7.1.1 (BioRad). Data from three independently stained gels of each sample were exported to Microsoft Excel for creation of the correction graphs, spot intensity graphs and statistical analysis.

The modified in-gel digestion method based on previous reports [25, 26] was performed for nanoelectrospray MS. Briefly, each spot of interest in the silver-stained gel was sliced and put into the microtube, and then washed twice with 50% ACN in 100 mM ammonium bicarbonate buffer (pH 8.0) for 10 min at room temperature. Subsequently, the excised-gel pieces were soaked in 100% ACN for 5 min, dried in a lyophilizer for 30 min and rehydrated in 50 mM ammonium bicarbonate buffer (pH 8.0) containing 10 mg/mL trypsin at 307C for 16 h. After digestion, the peptides were extracted from the supernatant of the gel elution solution (50% ACN in 5.0% TFA), and dried in a vacuum centrifuge.

The proteins were identified using an Ultimate capillary LC system (LC Packings, Amsterdam, The Netherlands) coupled to a QSTARXL quadrupole-time of flight (Q-TOF) mass spectrometer (Applied Biosystem/MDS Sciex, Foster City, CA, USA). The peptides was separated using an RP C18 capillary column (15 cm675 mm id) with a flow rate of 200 nL/min, and eluted with a linear ACN gradient from 10-50% ACN in 0.1% formic acid for 60 min. The eluted peptides from the capillary column were sprayed into the MS by a PicoTip electrospray tip (FS360-20-10-D-20; New Objective, Cambridge, MA, USA). Data acquisition from Q-TOF was performed using the automatic Information Dependent Acquisition (IDA; Applied Biosystem/MDS Sciex). Proteins were identified by the nanoLC-MS/MS spectra by searching against NCBI databases for exact matches using the ProID program (Applied Biosystem/MDS Sciex) and the MASCOT search program (http://www.matrixscience.com) [27] . A Homo sapiens taxonomy restriction was used and the mass tolerance of both precursor ion and fragment ions was set to 6 0.3 Da. Carbamidomethyl cysteine was set as a fixed modification, while serine, threonine, tyrosine phosphorylation and other modifications were set as variable modifications. The protein function and subcellular location were annotated using the Swiss-Prot (http://us.expasy.org/sprot/). The proteins were also categorized according to their biological process and pathway using the PANTHER classification system (http://www.pantherdb.org) as described in the previous studies [28] [29] [30] .

For determining subcellular localization, HL-CZ cells were transiently co-transfected with p3CLpro or pcDNA3.1 plus a mitochondrial localization vector pDsRed-Mito (Clontech) using the GenePorter reagent. After a 3-day incubation, the cells were fixed on glass coverslips with ice-cold acetone for 4 min, and blocked with 1% BSA. The co-transfected cells were subsequently incubated with mouse mAb anti-His tag, anti-cytochrome c, or rabbit anti-apoptosis-inducing factor (Sigma) at 47C overnight. After washing, the cells were incubated with FITC-conjugated goat anti-mouse immunoglobulin or anti-rabbit immunoglobulin at room temperature for 2 h. Confocal image analysis of the cells was performed using Leica TCS SP2 AOBS laser-scanning microscopy (Leica Microsystems, Heidelberg, Germany).

To identify specific cell responses to SARS CoV 3CLpro, the differential expression of proteins in mock cells and 3CLproexpressing cells were analyzed using 2-DE and nanoscale capillary LC/ESI Q-TOF MS. After confirming expression of SARS CoV 3CLpro in the transfected cells as previously described [16] , protein extracts prepared from mock cells and 3CLpro-expressing cells were separated using 2-DE. The resolved protein spots in gels were presented using silver staining ( Fig. 1 ). About 1000 protein spots in the pI range of 3.2 to 10 and the molecular weight range of 14 to 97.4 kDa were detected on the gels of mock ( Fig. 1A ) and 3CLproexpressing cells (Fig. 1B) , respectively. For comparison, three independent 2-DE images of each protein extract from three independent cell cultures of mock cells and 3CLpro-expressing cells were selected for statistical analysis. Protein profiling revealed that 154 6 15 up-regulated proteins and 141 6 12 down-regulated proteins in SARS CoV 3CLproexpressing cells were determined using GS-800 imaging densitometer with PDQuest software (Fig. 1 ). After the statistical analysis with Student's t-test, 75 up-regulated proteins (Spot ID number between 1 and 75) showed a statistically significant 1.5-fold increase in spot intensity (p,0.05) ( Table 1) , whereas 21 down-regulated proteins (Spot ID number between 76 and 96) had a statistically significant 2.0fold decrease in 3CLpro-expressing cells (Table 2) . Moreover, enlarged images of the selected protein spots were used to indicate spots with significant differences between mock cells and 3CLpro-expressing cells (Fig. 2) . A dramatic (greater than 100-fold) increase for Spot ID 19, a 3.3 6 0.13fold increase for Spot ID 55, a 5.29 6 0.12-fold decrease for Spot ID 83, and a 6.25 6 0.09-fold decrease for Spot ID 89 were found in 3CLpro-expressing cells (Fig. 2 , Tables 1 and Tables 1 and 2. 2). These selected protein spots were picked out of the stained gel, subjected to in-gel tryptic digestion, and underwent PMF using the NanoLC Trap Q-TOF MS (Tables 1 and  2 ). The representative peptide peaks from Q-TOF MS analysis were detected, such as 26S protease regulatory subunit 6A (Spot ID 21) (Fig. 3A) and apoptosis-inducing factor (Spot ID 55) (Fig. 3B) , resulting in confident protein identification by MASCOT searching. The search results indicated that 73 upregulated and 21 down-regulated proteins showed the best match with a protein score of greater than or equal to 67, considered to be significant using the MASCOT search algorithm (p,0.05) (Tables 1 and 2 ). The amino acid sequence coverage of identified up-regulatory and down-regulatory proteins varied from 9 to 85%. For example, ubiquitin-conjugating enzyme E2 N (Spot ID 24) had a MASCOT score of 217, sequence coverage of 52%, and eight matched peptides, while apoptosis-inducing factor (Spot ID 55) showed a MASCOT score of 252, sequence coverage of 18%, and eight matched peptides. Therefore, comparative analysis of protein profiling indicated that 73 up-regulated and 21 downregulated proteins were identified in 3CLpro-expressing cells.

As for the implication of cellular responses to SARS CoV 3CLpro, these up-regulated and down-regulated proteins were further categorized according to their subcellular location, biological process and biological pathway using the PANTHER classification system (Figs. 4 and 5 , Tables 1, 2  and 3) . Interestingly, up-regulated proteins in 3CLproexpressing cells were mainly located in the mitochondrion (26/73, 36%) (Fig. 4A) . By contrast, down-regulated proteins were distributed within different parts of the cells (19% in mitochondrion, 24% in cytoplasm, and 10% in nucleus) (Fig. 4B) . Biological process categorization revealed a diversity of biological processes associated with the proteins identified (Fig. 5) . The up-regulated proteins were responsible for the five main biological processes of protein metabolism and modification, nucleoside, nucleotide and nucleic acid metabolism, electron transport, pre-mRNA processing, and immunity and defense (Fig. 5 , Table 1 ). Comparison of the sub-categories of protein metabolism and modification showed significant differences between the biological process of up-regulated and down-regulated proteins (Fig. 5) .

The biological processes of proteolysis and protein modification were significantly up-regulated, but the biological processes of the protein biosynthesis and protein complex assembly were down-regulated in 3CLpro-expressing cells. Furthermore, 26S protease regulatory subunit 6A (Spot ID 21) and ubiquitin-conjugating enzyme E2 N (Spot ID 24), which is up-regulated in the biological processes of proteolysis and protein modification that are key to the ubiquitin proteasome pathway (Table 3 ). According to the biological pathway categorization, up-regulated proteins are associated with 11 signaling pathways, including de novo purine biosynthesis, ubiquitin proteasome, ATP synthesis, and apoptosis signaling pathways (Table 3) . Identified down-regulatory proteins in 3CLpro-expressing cells were involved in five signaling pathways, including de novo purine biosynthesis, apoptosis signaling, and mRNA splicing pathways (Table 3) . Interestingly, analysis of apoptosis signaling pathway revealed that the mitochondrial apoptogenic apoptosisinducing factor (Spot ID 55) was up-regulated and antiapoptogenic heat shock cognate 71-kDa protein (HSP70) (Spot ID 83) was down-regulated in 3CLpro-expressing cells (Table 3 ). This finding suggested that expression of SARS CoV 3CLpro resulted in activation of the apoptosis signaling pathway.

To confirm the expression levels of these identified proteins, Western blotting analysis of cell lysates from mock cells and SARS-CoV 3CLpro-expressing cells was carried out, in which beta-actin was used as an internal control (Fig. 6) . After normalization with beta-actin, densitometric analysis of immuno- reactive bands revealed that 26S protease regulatory subunit 6A and apoptosis-inducing factor were significantly increased 3-and 1.5-fold, respectively, in 3CLpro-expressing cells. The results were consistent with proteomic analyses of silver-stained 2-DE gels as shown in Fig. 1 .

To further investigate the role of mitochondria in SARS 3CLpro-induced apoptosis, confocal imaging analysis was applied to determine subcellular localization of apoptosisinducing factor (Fig. 7) . HL-CZ cells were transiently co-transfected with pcDNA3.1 or p3CLpro plus a mitochondrial localization vector pDsRed-Mito. After immunofluorescent staining, apoptosis-inducing factor labeled with FITC-conjugated secondary antibodies showed green fluoresce, whereas mitochondria were targeted by red fluorescent proteins (Fig. 7A) . Confocal imaging of the stained cells revealed that the release of apoptosis-inducing factor from mitochondria was found in the SARS CoV 3CLpro-expressing cells (Fig. 7A, right) , but not in mock cells (Fig. 7A, left) . In addition, cytochrome c, the other mitochondrial pro-apoptotic protein, was also found to be released from mitochondria to cytosol in 3CLpro-expressing cells (Fig. 7B) . The results indicated that SARS CoV 3CLpro induced mitochondria alternations in release of apoptosis-inducing factor and cytochrome c, therefore responsible for activation of upstream caspase-9 and downstream caspase-3.

In this study, 73 up-regulated and 21 down-regulated proteins in 3CLpro-expressing cells were identified using the combined analysis of 2-DE and Q-TOF MS ( Figs. 1 and 2 , Tables 1 and 2 ). Of the up-regulated proteins identified, 36% (26/73) were mitochondrial proteins that are associated with many biological processes, particularly in electron transport, ATP synthesis, carbohydrate metabolism and apoptosis (Fig. 4A , Table 1 ). By contrast, only 19% of down-regulated proteins were located within mitochondrion (Fig. 4B) . Up-regulation of activation of the mitochondrial electron transport system coupled with ATP synthesis was identified in SARS 3CLpro-expressing cells (Fig. 5, Tables 1 and 3 ), in which ATP synthase (Spot ID 19) and cytochrome c oxidase were also in involved in the control of mitochondrial membrane potential DCm and formation of reactive oxygen species (ROS) [31] . This finding could be associated with generation of ROS in 3CLpro-expressing cells as described in our previous report [16] . Protein metabolism and modification was the major biological process for up-regulated and down-regulated proteins in 3CLpro-expressing cells (Fig. 5) . However, analysis of the biological process indicated that proteolysis and protein modification were significantly up-regulated, but protein biosynthesis and protein complex assembly were downregulated in 3CLpro-expressing cells. Significant increases of Figure 5 . Comparison of biological processes associated with up-regulated and down-regulated proteins in 3CLpro-expressing cells compared with mock cells. Biological bioprocesses associated with up-and down-regulated proteins were classified using Panther Classification system (http://www.pantherdb.org/). Percent of biological process was calculated as the number of identified proteins in the indicated biological process/the number of the total identified proteins6100. the 26S protease regulatory subunit 6A (Spot ID 21) and ubiquitin-conjugating enzyme E2 N (Spot ID 24), which is involved in the ubiquitin proteasome pathway was demonstrated by sliver staining of 2-DE gels and Western blotting (Figs. 1, 2 and 6 ). Interestingly, up-regulation of proteasome subunits was also found in SARS CoV-infected Vero E6 cells and hepatitis virus B (HBV) HBx-expressing mice [20, 22] . The ubiquitin-proteasome pathway plays a central role in several cellular processes including antigen processing, apoptosis, cell cycle, inflammation, and response to stress [32] , and is involved in the replication of several viruses, such as mouse hepatitis virus (murine coronavirus) [33, 34] , adenovirus [35] , hepatitis C virus [36] , and human immunodeficiency virus [37] . Therefore, up-regulation of the ubiquitin-proteasome pathway induced by SARS-CoV 3CLpro protein might be involved in the SARS pathogenesis. Five kinds (C1/C2, A/B, D0, A2/B1 and M) of heterogeneous nuclear ribonucleoproteins (hnRNP) were identified as being significantly up-regulated in SARS-CoV 3CLpro-expressing cells (Fig. 1, Table 1 ), and responsible for transcription, pre-mRNA processing, mRNA splicing, and nucleoside, nucleotide and nucleic acid metabolism ( Fig. 5 and Table 1 ). The finding was in agreement with the protein profiling of SARS-CoV-infected cells in a previous report [20] . HnRNP have also been reported to be involved in the replication of mouse hepatitis virus (MHV) [38, 39] . In addition, hnRNP A2/B1 and A/B have been demonstrated to interact with the negative-strand MHV leader RNA and to enhance Figure 6 . Western blot analysis of 26S protease regulatory subunit 6A and apoptosis-inducing factor in mock cells and 3CLproexpressing cells. Each lysate was analyzed by 12% SDS-PAGE, and then electrophorectically transferred onto NC paper. The blot was probed with monoclonal antibodies to 26S protease regulatory subunit 6A and apoptosis-inducing factor, and developed with an alkaline phosphatase-conjugated secondary antibody and NBT/BCIP substrates. Lane 1: mock cells; lanes 2: 3CLproexpressing cells. MHV RNA synthesis [40] . Therefore, the SARS-CoV 3CLpro that induced a significant increase in hnRNP expression could play an important role in coronavirus infection.

Interestingly, analysis of the apoptosis signaling pathway revealed that the mitochondrial apoptogenic apoptosisinducing factor (Spot ID 55) was up-regulated and antiapoptogenic heat shock cognate 71-kDa protein (HSP70) (Spot ID 83) was down-regulated in 3CLpro-expressing cells (Figs. 1, 2 and 6, Tables 1, 2 and 3) . Apoptosis-inducing factor (Spot ID 55) was also released from the mitochondria of 3CLpro-expressing cells (Fig. 7A) . Release of apoptosis-inducing factor from mitochondria results in a caspase-independent pathway of programmed cell death [41, 42] . Moreover, the other mitochondrial apoptogenic protein cytochrome c has been demonstrated to be released from mitochondria (Fig. 7B) . Since the activation of caspase-3 and caspase-9 has been demonstrated previously to be involved in 3CLpro-inducing apoptosis [16] , release of apoptosis-inducing factor and cytochrome c in the cytosol ise suggested to interact with apoptotic protease-activating factor 1 (Apaf-1) and dATP/ATP to activate apoptosomemediated caspase-9 and process downstream effector caspases such as caspase-3. Therefore, the results reveal that SARS CoV 3CLpro induces mitochondrial-mediated apoptosis.

In conclusion, proteomic analysis of cellular responses to SARS CoV 3CLpro demonstrated that SARS CoV 3CLpro up-regulates the main biological process of protein metabolism and modification, particularly in the ubiquitin proteasome pathway. Moreover, the results showed that SARS CoV 3CLpro induces mitochondrial alternation in apoptosis signaling, electron transport and ATP synthesis. The study provides system-level insights into the interaction of SARS CoV 3CLpro with host cells, and is helpful for elucidating the molecular basis of SARS CoV pathogenesis.

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We would like to thank China Medical University and National Science Council, Taiwan for financial supports (CMU95-152, CMU95-153, NSC94-2320-B-039-010 and  NSC95-2320-B-039-019) .

De novo purine biosynthesis 25 Nucleoside