【病毒外文文獻(xiàn)】2008 Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP

Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation Kartika Padhan Rinki Minakshi Mohammad Aatif Bin Towheed and Shahid Jameel Correspondence Shahid Jameel shahid icgeb res in Virology Group International Centre for Genetic Engineering and Biotechnology Aruna Asaf Ali Marg New Delhi 110 067 India Received 17 December 2007 Accepted 11 April 2008 The molecular mechanisms governing severe acute respiratory syndrome coronavirus induced pathology are not fully understood Virus infection and some individual viral proteins including the 3a protein induce apoptosis However the cellular targets leading to 3a protein mediated apoptosis have not been fully characterized This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways Activation of caspase 8 through extrinsic signal s caused Bid activation In the intrinsic pathway there was activation of caspase 9 and cytochrome c release from the mitochondria This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein expressing cells which depended on the activation of p38 MAP kinase MAPK in these cells For p38 activation and apoptosis induction the 3a cytoplasmic domain was sufficient In direct Annexin V staining assays the 3a protein expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580 A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed These results have been used to present a model of 3a mediated apoptosis INTRODUCTION The aetiological agent for severe acute respiratory syn drome SARS was identified as a novel coronavirus SARS CoV Peiris et al 2003 SARS CoV has a polyadenylated positive sense RNA genome of approxi mately 30 kb Marra et al 2003 In addition to the prototypic coronavirus genes the SARS CoV genome also contains nine unique open reading frames ORFs Marra et al 2003 Of these orf3a is the largest and encodes a protein of 274 aa variously called ORF3A Ito et al 2005 X1 Rota et al 2003 or U274 Tan et al 2004b The 3a protein has been predicted to contain an N terminal signal peptide followed by three transmembrane domains and a C terminal cytoplasmic domain of approximately 150 aa Zeng et al 2004 The 3a protein is associated with virus particles produced following infection of Vero E6 or Caco 2 cells Ito et al 2005 Shen et al 2005 and can assemble into virus like particles when co expressed with the membrane and envelope proteins in insect cells Shen et al 2005 In vitro studies have also shown the 3a protein to interact with the viral envelope membrane and spike proteins Tan et al 2004b Tan 2005 and the cellular protein caveolin 1 Padhan et al 2007 A deletion of orf3a was shown to reduce virus titres but not to eliminate virus replication Yount et al 2005 and convalescent sera from SARS patients have antibodies to the 3a protein Tan et al 2004a suggesting that it is expressed during virus infection of the host The 3a protein was shown to upregulate the expression of fibrinogen in A549 lung epithelial cells Tan et al 2005 and to possess an ion channel activity selective for monovalent cations Lu et al 2006 Ectopic expression of the 3a protein has been shown to induce apoptosis in Vero E6 cells through activation of caspase 8 chromatin condensation and DNA fragmenta tion Law et al 2005 In a Drosophila melanogaster expression system it was also found to modulate cellular cytochrome c levels and caspase activity Wong et al 2005 It has recently been shown to induce G 1 phase cell cycle arrest via the cyclin D3 pRb pathway in HEK293 COS 7 and Vero cells Yuan et al 2007 Apoptosis is a cell death programme that is initiated in response to various extrinsic and intrinsic signals such as cellular stress including virus infection and plays an important role in the pathogenesis of many viruses Wang et al 2007 The extrinsic pathway initiates at the cell surface following occupation of death receptors by their cognate ligands the best examples of this are the Fas Fas ligand and tumour necrosis factor receptor TNFR TNF Journal of General Virology 2008 89 1960 1969 DOI 10 1099 vir 0 83665 0 1960 0008 3665 G 2008 SGM Printed in Great Britain pathways Tartaglia et al 1993 The intrinsic pathway is controlled by multiple sensor proteins such as those of the Bcl 2 family and initiates at the mitochondria leading to their depolarization and leakage of molecules such as cytochrome c Green Oda et al 2000 This is either through association with anti apoptotic Bcl 2 family members or by stimulating other apoptosis promoting factors Chen et al 2005 Many apoptotic stimuli utilize Bax to kill cells Zhang et al 2000 Its overexpression alone is also sufficient to induce apoptosis in many cell types Xiang et al 2000 The tumour suppressor and transcription factor p53 can also enhance the pro apoptotic activity of Bax either by inducing its expression or by sequestrating Bcl 2 or Bcl X L in the cytoplasm Miyashita Sigma at 30 uC for 30 min and the reaction stopped with 50 mM Tris HCl pH 8 0 The lysate was then prepared in 26 in SDS loading buffer boiled and separated by 10 SDS PAGE Immunofluorescence and subcellular localization Cells grown on coverslips to 40 50 confluence were transfected and then imaged as described previously Padhan et al 2007 For subcellular localization of cytochrome c Huh7 cells were co transfected with pEGFP cytC and pDsRed mito Clontech either with or without plasmid 3a ECFP The percentage co localization was calculated using LaserPix software Bio Rad For STAT3 imaging Huh7 cells were co transfected with the STAT3 EGFP vector together with either the 3a DsRed or the pDsRed N1 plasmid After 36 h cells were serum starved overnight and then treated with 100 ng epidermal growth factor EGF ml 21 for 3 h washed with PBS and imaged Flow cytometry Huh7 cells were transfected as above and 36 h later the cells were switched to fresh medium containing 20 mM SB203580 for 12 h Subsequently 1 5610 6 1 8610 6 cells were harvested in 0 02 EDTA washed twice with PBS and stained with 3 ml phycoerythrin PE conjugated Annexin V BD Pharmingen in 40 ml binding buffer 10 mM HEPES pH 7 4 140 mM NaCl 2 5 mM CaCl 2 The cells were then fixed with 100 ml fixative Intrastain kit Dako for 15 min at room temperature and washed with PBS The pellet was resuspended in 100 ml permeabilization buffer Intrastain kit Dako at room temperature for 15 min After washing with PBS the cells were resuspended in 1 100 diluted rabbit anti 3a and incubated for 30 min at room temperature followed by two washes in PBS The cells were then counterstained with 1 500 diluted Alexa 488 conjugated anti rabbit antibody Molecular Probes at room temperature for 45 min After two washes in PBS containing 1 BSA the cells were resuspended in 400 ml of the same buffer for acquisition For each sample 30000 events were acquired using a CyAn ADP flow cytometer Dako and the data analysed using Summit software version 4 3 The cells were gated for 3a expression and Annexin V staining was determined in 3a positive and negative cells the latter being treated as controls Apoptosis SD was calculated over four different experiments and the results SARS CoV 3a protein and apoptosis http vir sgmjournals org 1961 were calculated as percentage values P values were calculated using Student s t test with 95 confidence intervals Luciferase assay Transfected cells were collected at 36 h p t serum starved overnight and then treated with 100 ng EGF ml 21 for 3 h washed with PBS and harvested The preparation of cytosolic extracts and luciferase assays were carried out using a Luciferase assay kit Promega according to the supplier s protocol and readings were taken on a luminometer Sirius RESULTS SARS CoV 3a protein activates the intrinsic pathway of apoptosis The 3a protein has been shown previously to induce apoptosis in Vero E6 cells In this system there was increased caspase 8 activation but no change in Bad or Bcl 2 levels suggesting that the death receptor extrinsic apoptosis pathway was activated Law et al 2005 We chose to study the apoptotic activity of the 3a protein in the Huh7 human hepatocellular carcinoma cell line as liver cells have been shown to be permissive for SARS CoV infection and replication Kaye 2006 Furthermore these cells are negative for caveolin unlike Vero or A549 cells Damm et al 2005 We recently showed that the SARS CoV 3a protein interacts with caveolin Padhan et al 2007 a cellular protein with multiple effects including effects on apoptosis Timme et al 2000 Huh7 cells transfected with the pSGI 3a HA or control plasmid were harvested at different times and checked for various cell death markers by Western blotting There was increased activation cleavage of caspase 3 the central effector caspase and its downstream substrates caspase 7 and PARP in 3a protein expressing cells Fig 1a Caspase 3 can be activated by either caspase 8 from the extrinsic pathway or caspase 9 from the intrinsic pathway Higher levels of cleaved caspase 9 were detected in 3a protein expressing cells these effects being more prominent following serum starvation Fig 1b As the 3a protein has been shown previously to activate caspase 8 Law et al 2005 we also checked for cleavage of Bid Truncated Bid tBid has effects on the mitochondria and is important for cross talk between the extrinsic and intrinsic pathways Luo et al 1998 Higher levels of tBid were observed in 3a protein expressing cells Fig 1c Another hallmark of the intrinsic pathway of apoptosis is the release of cytochrome c from mitochondria In Huh7 cells co transfected with pEGFP cytC and pDsRed mito the cytochrome c signal was found quantitatively in the mitochondria in the absence of 3a protein Fig 2 upper panels However in cells that co expressed 3a cytochrome c was found to be extra mitochondrial Fig 2 middle panels The 3a protein itself did not localize to the mitochondria Fig 2 lower panels and was found predominantly at the plasma membrane and in the Golgi region as shown previously Padhan et al 2007 On image quantification across multiple cells that expressed the 3a protein only 7 1 4 0 of the cytochrome c signal was found to co localize with the mitochondrial marker Thus caspase 9 activation and cytochrome c release in cells expressing the 3a protein indicated that this viral protein also activates the intrinsic pathway of apoptosis Bax oligomerization and p53 upregulation are promoted by the 3a protein Members of the Bcl 2 family of proteins play a major role in regulating cytochrome c release from the mitochondria There were no significant differences in the levels of Bak Bcl X L Bcl 2 and Mcl 1 as well as the outer mitochondrial a b c Fig 1 Apoptosis markers in 3a protein expressing cells a Huh7 cells were trans fected with pSG 3a or the control vector pSGI At various times cell lysates were analysed by Western blotting for the indicated apoptosis marker proteins b Transfected Huh7 cells were either shifted to serum free medium at 36 h p t for 12 h or kept in serum containing medium Cell lysates were analysed by Western blotting c Transfected Huh7 cells at 48 h p t were analysed by Western blotting K Padhan and others 1962 Journal of General Virology 89 membrane porin VDAC in 3a protein expressing versus control cells Fig 3a However cells expressing the 3a protein showed increased levels of oligomeric Bax Fig 3b These levels also varied with time peaking at 48 h following pSGI 3a HA transfection of Huh7 cells Oligomeric Bax forms a channel in the outer mitochon drial membrane through which cytochrome c and other apoptogenic factors move into the cytosol The tumour suppressor protein p53 is pro apoptotic and works through diverse mechanisms One of these is based on the activation of bax transcription as the p53 mediated activation of Bax at cytoplasmic sites Baptiste this would also contribute to PARP cleavage but would be insensitive to SB203580 The activation of p38 is regulated by various stress signals and is mediated by the upstream MAPK kinase isoforms 3 and 6 MKK3 6 Increased levels of phosphorylated activated MKK3 6 were also observed in 3a protein expressing cells Fig 4d Taken together these results support a stress pathway mediated p38 activation that is directly responsible for p53 upregulation and Bax oligo merization in 3a protein expressing cells The cytoplasmic domain of the 3a protein but not its caveolin binding activity is required for apoptotic activity The 3a protein of SARS CoV is predicted to contain three transmembrane regions aa 34 56 77 99 and 103 125 and a C terminal cytoplasmic domain of 149 aa Expression of the 3a cytoplasmic domain in Huh7 cells led to increases in PARP cleavage and p38 phosphorylation at levels similar to those observed with the full length protein Fig 5a On densitometric quantification cells transfected with pSG 3a or pSG 3a Cyto showed a mean 2 3 and 2 1 fold increase in cleaved PARP respectively compared with vector transfected cells This indicated that the 3a cytoplasmic domain was sufficient for its pro apoptotic effects We showed recently that the 3a protein interacts with caveolin a protein that regulates many cellular processes including apoptosis Padhan et al 2007 As the analysis thus far was in caveolin null Huh7 cells we also tested the pro apoptotic effects of the 3a protein in caveolin expressing monkey kidney COS cells Immunofluorescent staining of caveolin and 3a protein was carried out in cells transfected with pSG 3a This confirmed that Huh7 cells did not express caveolin whilst COS cells did and that both cells showed a similar intracellular distribution of ectopi cally expressed 3a protein Fig 5b Western blotting of COS cell lysates showed that in this cellular background the 3a protein also induced PARP cleavage and p38 MAPK activation Fig 5b Other elements of the 3a protein mediated apoptotic pathway such as Bax oligomerization p53 upregulation and caspase 9 activation were also observed in COS cells Fig 5c These results suggested that the pro apoptotic nature of the 3a protein is not influenced by its ability to bind caveolin Activation of p38 MAPK is important for 3a protein mediated apoptosis The results reported thus far indirectly suggested a pro apoptotic role for the 3a protein To demonstrate directly an effect of the 3a protein on cell death we measured the ability of 3a protein expressing and control cells to bind Annexin V An early event in apoptosis is the loss of membrane phospholipid asymmetry resulting in the exposure of phosphatidylserine at the surface of the cell Annexin V binds with high affinity to phosphatidylserine and this binding is used as a marker of apoptosis Koopman et al 1994 Using flow cytometry we measured Annexin V binding to Huh7 cells that expressed the 3a protein and compared this with control cells that did not express the protein There was a significant increase in Annexin V binding to cells that expressed the 3a protein a b c d Fig 4 The 3a protein mediated p53 upregulation and Bax dimerization are p38 dependent a Huh7 cells at 48 h p t were analysed for various MAPKs by Western blotting b Transfected Huh7 cells at 36 h p t were shifted to fresh medium without mock or with 20 mM SB203580 After 12 h cell lysates were prepared and analysed by Western blotting for the indicated proteins c Lysates prepared from transfected Huh7 cells either treated with SB203580 or not were subjected to cross linking with DSS as described in Methods The lysates were then Western blotted for Bax d Lysates from Huh7 cells at 48 h p t were analysed for pMKK3 6 by Western blotting K Padhan and others 1964 Journal of General Virology 89 compared with control cells Fig 6a However treatment of the 3a protein expressing cells with the p38 MAPK inhibitor SB203580 led to reduced Annexin V binding Fig 6a In four separate experiments we observed a mean of approximately 40 of cells expressing the 3a protein undergoing apoptosis compared with 0 5 of control cells Treatment with SB203580 reduced this to about half with a mean of 19 of 3a expressing cells demonstrating apoptosis Fig 6b This indicated that p38 MAPK activation is important for the apoptosis of 3a protein expressing cells The 3a protein inhibits nuclear translocation of STAT3 STAT3 is a key factor that negatively regulates p53 transcription by binding to its promoter Niu et al 2005 We checked the subcellular localization of trans fected STAT3 EGFP either alone or in cells expressing the 3a protein In control cells following EGF stimulation most of the STAT3 was found in the nuclear compartment Fig 7a i However in 3a protein expressing cells under similar conditions of EGF stimulation STAT3 was retained in the cytoplasm showing a punctate distribution and co localization with the 3a protein Fig 7a ii iv We tested whether the 3a protein binds STAT3 to retain it in the cytoplasm In a co immunoprecipitation assay no direct interaction was detected between these two proteins data not shown To confirm further the inhibition of STAT3 nuclear translocation we used a reporter gene transfection assay in which plasmid pLucTKS3 containing a minimal thymidine kinase promoter and seven STAT3 response elements was used to drive luciferase expression Transiently transfected Huh7 cells showed a marked reduction in luciferase activity when pLucTKS3 was co transfected with the pSG 3a expression plasmid compared with the vector control Fig 7b Furthermore by Western blotting no significant differences were observed in the levels of phospho STAT3 and total STAT3 in control versus 3a protein expressing cells Fig 7c Taken together these results showed that in cells that express the SARS CoV 3a protein there is reduced nuclear localization and consequently decreased transcription factor activity of STAT3 DISCUSSION The host uses apoptosis to limit virus replication and remove virus infected cells Fujimoto et al 2000 Apoptosis can also assist in the dissemination of viral particles and is the cause of tissue damage observed following many viral infections Lyles 2000 In SARS patients apoptosis of cells of the lung epithelium has been observed suggesting SARS CoV induced apoptosis to have a deleterious pathogenic role leading to severe tissue damage Yan et al 2004 There are indications that apoptosis and the antiviral immune response may both contribute to the pathology in SARS patients Yang et al 2005 The SARS CoV 3a 3b nucleocapsid and 7a proteins have been shown to induce apoptosis Law et al 2005 Yuan et al 2007 In Vero E6 cells that transiently express the 3a protein nuclear fragmentation chromatin condensation DNA laddering and an increased terminal deoxynucleoti dyl transferase dUTP nick end labelling signal have been a b c Fig 5 a Huh7 cells were transfected with expression plasmids for full length or the cytoplasmic domain of 3a protein Lysates prepared at 48 h p t were analysed by Western blotting b Left panels Huh7 and COS cells transfected with pSG 3a were fixed permeabilized and stained with antibod ies against 3a protein or caveolin 1 followed by confocal microscopy Right panels lysates prepared from cells at 48 h p t were analysed by Western blotting against the indicated proteins c Lysates of transfected COS cells at 48 h p t were cross linked with DSS and Western blotted for Bax Cell lysates without cross linking were analysed for p53 and caspase 9 levels SARS CoV 3a protein and apoptosis http vir sgmjournals org 1965 observed Law et al 2005 Furthermore there was increased caspase 8 activation but no change in Bcl 2 Bad or PCNA levels Law et al 2005 As caspase 8 is downstream of the cell surface death receptors Green this results in the release of cytochrome c and initiation of apoptosis Wolter et al 1997 What might be the cue for Bax oligomerization Whilst there are many cellular signals that regulate Bax le