《【病毒外文文獻(xiàn)】2007 Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interact》由會(huì)員分享，可在線閱讀，更多相關(guān)《【病毒外文文獻(xiàn)】2007 Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interact（10頁(yè)珍藏版）》請(qǐng)?jiān)谘b配圖網(wǎng)上搜索。

JOURNAL OF VIROLOGY June 2007 p 6346 6355 Vol 81 No 12 0022 538X 07 08 00H110010 doi 10 1128 JVI 00090 07 Copyright 2007 American Society for Microbiology All Rights Reserved Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl X L Protein H17188 Ying Xim Tan 1 Timothy H P Tan 1 Marvin J R Lee 1 Puay Yoke Tham 1 Vithiagaran Gunalan 1 Julian Druce 2 Chris Birch 2 Mike Catton 2 Nai Yang Fu 3 Victor C Yu 3 and Yee Joo Tan 1 Collaborative Anti Viral Research Group Institute of Molecular and Cell Biology Republic of Singapore 1 Victorian Infectious Diseases Reference Laboratory North Melbourne Victoria Australia 2 and Mechanisms of Apoptosis in Mammalian Cells Group Institute of Molecular and Cell Biology Republic of Singapore 3 Received 14 January 2007 Accepted 23 March 2007 The severe acute respiratory syndrome coronavirus SARS CoV 7a protein which is not expressed by other known coronaviruses can induce apoptosis in various cell lines In this study we show that the overexpression of Bcl X L a prosurvival member of the Bcl 2 family blocks 7a induced apoptosis suggesting that the mech anism for apoptosis induction by 7a is at the level of or upstream from the Bcl 2 family Coimmunoprecipi tation experiments showed that 7a interacts with Bcl X L and other prosurvival proteins Bcl 2 Bcl w Mcl 1 and A1 but not with the proapoptotic proteins Bax Bak Bad and Bid A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl X L was observed suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl X L Interestingly amino acids 224 and 225 within the C terminal transmembrane domain of Bcl X L are essential for the interaction with the 7a protein although the BH3 domain of Bcl X L also contributes to this interaction In addition fractionation experiments showed that 7a colocalized with Bcl X L at the endoplasmic reticulum as well as the mitochondria suggesting that they may form complexes in different membranous compartments Many virus genomes encode gene products that can modu late apoptosis also called programmed cell death and the regulation of apoptosis in the infected host is an important determinant in the struggle between virus and host for survival In the case of the severe acute respiratory syndrome corona virus SARS CoV one of the most common abnormalities in infected patients is lymphopenia which is caused by the de pletion of T lymphocytes by apoptosis 4 5 22 In addition apoptosis was observed in various infected tissues such as lung liver and thyroid obtained during autopsy studies on SARS casualties 3 32 Interestingly necrosis another form of cell death was also observed in different tissues obtained from SARS CoV infected patients 6 9 17 Taken together these observations suggest that the modulation of cell death during SARS CoV infection is important for viral replication and or pathogenesis The family of Bcl 2 related proteins constitutes one of the biologically important gene products in the process of apopto sis Several members of this family are prosurvival as their overexpression inhibits apoptosis induced by many different stimuli while other members are proapoptotic as their over expression promotes apoptosis 2 34 Besides playing impor tant roles in regulating apoptosis and maintaining homeostasis in healthy cells members of the Bcl 2 family are also impli cated in viral infections Indeed some viruses encode proteins that are functional homologues of members of the Bcl 2 family or are capable of interacting with members of the Bcl 2 family 8 12 23 33 Interestingly it was recently demonstrated that the prosurvival Bcl 2 protein inhibits the caspase dependent apoptosis induced by SARS CoV infection without affecting viral replication kinetics 1 In addition it has been shown that the SARS CoV envelope protein induces apoptosis in Jurkat T cells in the absence of growth factors and that this event could be inhibited by the overexpression of Bcl X L another prosur vival member of the Bcl 2 family 37 The SARS CoV 7a protein which shows no significant se quence homology to viral proteins of other known coronavi ruses can induce caspase dependent apoptosis in cell lines derived from different organs including the lung kidney and liver 16 28 In this study we show that the overexpression of Bcl X L blocks the induction of apoptosis by 7a suggesting that 7a may be acting at the level of or upstream from the Bcl 2 family A detailed analysis of the properties of 7a with respect to its ability to interact with different members of the Bcl 2 family and its cellular localization suggests that 7a induces apoptosis by interfering directly with the function of the pro survival proteins like Bcl X L Bcl w Mcl 1 and A1 MATERIALS AND METHODS Materials All reagents used in this study were purchased from Sigma St Louis MO unless otherwise stated All cell lines were purchased from the American Type Culture Collection Manassas VA and cultured at 37 C in 5 CO 2 in Dulbecco s modified Eagle s medium containing 1 g liter glucose 2 mM liter glutamine 1 5 g liter sodium bicarbonate 0 1 mM nonessential amino acids 0 1 mg ml streptomycin 100 U penicillin and 5 fetal bovine serum HyClone Construction of plasmids Expression plasmids for 7a and Bcl X L deletion and substitution mutants were generated by PCR using titanium Taq DNA polymer ase Clontech Laboratories Inc Palo Alto CA All sequences were confirmed Corresponding author Mailing address Institute of Molecular and Cell Biology 61 Biopolis Drive Proteos Singapore 138673 Re public of Singapore Phone 65 65869625 Fax 65 67791117 E mail mcbtanyj imcb a star edu sg Y X T and T H P T contributed equally to this work H17188 Published ahead of print on 11 April 2007 6346 on March 9 2015 by WEST VIRGINIA UNIV http jvi asm org Downloaded from by sequencing performed by the core facilities at the Institute of Molecular and Cell Biology Singapore All primers used in this study were purchased from Research Biolabs Singapore Generation of stable cell lines The Bcl X L open reading frame ORF was cloned into the pCep4 vector Invitrogen Carlsbad CA and transfected into 293T cells by electroporation as previously described 30 Cells stably expressing Bcl X L were obtained after selection in 0 2 mg ml of hygromycin B Roche Molecular Biochemicals Indianapolis IN Control cells were stably transfected with an empty vector Transient transfections CaspACE fluorometric assay and Western blot anal ysis Transient transfections of 293T and Vero E6 cells were performed using Lipofectamine reagent Invitrogen according to the manufacturer s protocol Approximately 16 h after transfection the activation of caspase 3 was quantified by using a CaspACE fluorometric assay system from Promega Corporation Madison WI as previously described 15 28 Western blot analysis was performed as previously described 31 and some of the primary antibodies anti myc monoclonal Santa Cruz Biotechnology Santa Cruz CA anti SARS CoV M rabbit polyclonal Abgent San Diego CA anti actin and anti tubulin monoclonal Sigma anti pyruvate dehydrogenase PDH E2 subunit Molecular Probes OR anti Bcl X L monoclonal Transduction Lab oratories anti BAD rabbit polyclonal Cell Signaling Technology Inc Beverly MA and anti calreticulin and anti alpha COP I rabbit polyclonal Affinity BioReagents CO antibodies were purchased while an anti 7a monoclonal antibody was generated for this study Briefly a bacterially expressed glutathione S transferase GST 7a fusion protein was used to immunize BALB c mice as previously described 10 The spleen was excised from a mouse that showed strong antibody response and hybridoma fusion was performed to generate anti 7a monoclonal antibodies as previously described 20 All procedures in volving the use of laboratory animals were performed by trained personnel in accordance with the regulations and guidelines of the National Advisory Com mittee for Laboratory Animal Research NACLAR Singapore The other an tibodies against SARS CoV proteins anti N and anti 3a mouse polyclonal an tibodies have been described previously 11 31 Coimmunoprecipitation experiments For the coimmunoprecipitation experi ments each 6 cm dish of cells was resuspended in 200 H9262l of immunoprecipitation IP buffer 50 mM Tris pH 8 150 mM NaCl 0 5 NP 40 0 5 deoxycholic acid 0 005 sodium dodecyl sulfate SDS and 1 mM phenylmethylsulfonyl fluoride and subjected to freeze thawing six times One hundred microliters of the lysates was diluted with 50 H9262l of IP buffer 5 H9262l of rabbit anti myc polyclonal antibody Santa Cruz Biotechnology was added and the mixture was subjected to end over end mixing at room temperature for 1 h Protein A agarose beads Roche were then added and the mixing was continued for at least4hat4 C Beads were washed four times with cold IP buffer and then 15 H9262l of Laemmli s SDS buffer was added and the samples were boiled at 100 C for 5 min to release the immunocomplexes Samples were separated by SDS polyacrylamide gel elec trophoresis and subjected to Western blot analysis Coimmunoprecipitation experiments with SARS CoV infected cells were per formed in a similar manner Infection of Vero E6 cells with an isolate of SARS CoV strain HKU 39849 was carried out in a physical containment level 4 laboratory as previously described 14 Lysates obtained from SARS CoV infected Vero E6 cells were subjected to Western blot analysis to determine the expression levels of different viral proteins or to IP with either rabbit anti Bcl X L polyclonal antibody Santa Cruz Biotechnology or an irrelevant antibody anti BID rabbit polyclonal antibody Santa Cruz Biotechnology and protein A aga rose beads Western blot analyses were then performed to detect the amounts of 7a or Bcl X L in the immunocomplexes captured on the protein A agarose beads Fractionation experiments Intact mitochondria were isolated from 293T cells expressing 7a by using a mitochondrial isolation kit for cultured cells from Pierce Rockford IL The reagent based method was used according to the manufac turer s protocol and this method gave two fractions The first fraction contained nonmitochondrial proteins while the second fraction contained only mitochon drial proteins Both fractions were made up to equal volumes 500 H9262l H110118 H11003 10 6 cells and subjected to Western blot analysis as described above The same protocol was used to fractionate SARS CoV infected Vero E6 cells that were harvested at 0 16 or 24 h postinfection Indirect immunofluorescence experiments Vero E6 cells were transfected with cDNA for expressing 7a as described above After 16 h the cells were incubated with 50 nM of MitroTracker red Molecular Probes Inc Eugene OR for 15 min at 37 C Then the cells were processed for antibody staining as previously described 31 RESULTS Overexpression of Bcl X L inhibits the induction of apopto sis by the SARS CoV 7a protein In order to determine if the SARS CoV 7a protein acts upstream or downstream of the Bcl 2 family to induce apoptosis 293T cells stably expressing Bcl X L were generated As shown in Fig 1A there is high expression of Bcl X L in the chosen stable clone 293T Bcl X L in comparison to the endogenous Bcl X L level in the vector control cells 293T Vec These stable cell lines were then transiently transfected with cDNA to express HA BAX posi tive control or 7a and the induction of apoptosis was deter mined as previously described 28 The expression of HA BAX and 7a caused apoptosis in 293T Vec cells Fig 1B columns 1 to 4 as determined by a fluorometric assay for measuring caspase 3 activation However in the 293T Bcl X L cells the expression of HA BAX and 7a did not induce significant levels of apoptosis Fig 1B columns 5 to 8 Thus the overexpression of Bcl X L inhibited the 7a induced apoptosis suggesting that the regulation of cellular apopto sis by 7a occurs at or upstream of the Bcl 2 family in the apoptotic pathway Previously we constructed a 7a cDNA expression plasmid MG 7a with six additional base pairs ATGGGA which code for two additional amino acids methionine and glycine before the ATG codon of the 7a ORF in order to achieve a Kozak consensus ribosome binding site for efficient translation initiation 10 Here a new 7a cDNA expression plasmid with out the Kozak sequence was constructed As shown in Fig 1C the cleavage of the signal peptide of the 7a protein is more efficient than that of the protein expressed using the old cDNA expression plasmid compare lanes 3 and 4 or lanes 7 and 8 However there is no difference in the degree of apoptosis induced by the transfection of either construct which is con sistent with our previous finding that the cleavage of the signal peptide of 7a is not important for the induction of apoptosis 28 The 7a construct without the Kozak sequence was used for the subsequent studies SARS CoV 7a interacts with Bcl X L but not with proapop totic members of the Bcl 2 family To further understand how the SARS CoV 7a protein modulates the function of the Bcl 2 family of proteins we performed coimmunoprecipitation ex periments to determine if 7a can interact with members of this family of proteins As shown in Fig 2A top panel 7a was specifically coimmunoprecipitated by myc Bcl X L lane 2 but not by the negative control myc GST lane 1 or the proapop totic proteins myc BAD myc BID myc BAX or myc BAK tested lanes 3 to 6 These results indicate that 7a may induce apoptosis by interfering with the prosurvival function of Bcl X L As 293T cells do not support SARS CoV replication we determined the temporal expression of 7a in infected Vero E6 an African green monkey kidney cell line The expression of 7a and other SARS CoV proteins 3a M and N was detected in cells harvested at 16 h and 24 h postinfection Fig 2B Co immunoprecipitation experiments were then performed using lysates obtained at 24 h postinfection As shown in Fig 2C 7a was coimmunoprecipitated by an anti Bcl X L antibody and not by an anti BID antibody suggesting that the 7a and endoge nous Bcl X L proteins formed complexes during SARS CoV VOL 81 2007 INTERACTION BETWEEN SARS CoV 7a PROTEIN AND Bcl X L 6347 on March 9 2015 by WEST VIRGINIA UNIV http jvi asm org Downloaded from FIG 1 Effects of Bcl X L on the induction of apoptosis by the SARS CoV 7a protein A Western blot analysis was performed to determine the expression level of Bcl X L in the stable cell line 293T Bcl X L compared to the expression level in the vector control 293T Vec cells Equal amounts of cells were used in each lane as verified by the levels of endogenous actin Molecular mass markers are shown on the left B A CaspACE fluorometric assay system from Promega Corporation Madison WI was used to measure the activation of caspase 3 protease activity which is a hallmark of apoptosis in cells that were transfected with vector only a classical apoptosis inducer HA BAX 7a with two additional amino acids at its N terminus MG 7a or full length 7a The cells used were 293T Vec or 293T Bcl X L All experiments were performed in duplicate and the average values with standard deviations are plotted C A corresponding Western blot analysis was performed to determine the expression levels of 7a HA BAX and Bcl X L using anti 7a anti HA and anti Bcl X L antibodies respectively The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin anti actin Molecular mass markers are shown on the left 6348 TAN ET AL J VIROL on March 9 2015 by WEST VIRGINIA UNIV http jvi asm org Downloaded from infection This experiment was performed three times and the data presented in Fig 2C are a representative set The transmembrane domain of the SARS CoV 7a protein is important for its interaction with Bcl X L and for the induction of apoptosis Next we constructed a series of 7a mutants and compared their abilities to induce apoptosis The C terminal portion of the 7a protein contains a transmembrane domain 98 to 116 amino acids aa and a short cytoplasmic tail 117 to 122 aa 10 While the transmembrane domain is important for the insertion of 7a into intracellular membranes the cyto plasmic tail contains an endoplasmic reticulum ER retrieval motif that mediates the transport of 7a between the Golgi and the ER As demonstrated by the activation of caspase 3 Fig 3A the 7a 1 116 aa deletion mutant columns 3 and 8 induced as much apoptosis as the full length 7a indicating that the cytoplasmic tail of 7a is not essential for apoptosis induc tion However the 7a 1 97aa and 7aH900498 116aa deletion mu tants columns 4 5 9 and 10 did not induce significant levels of apoptosis in either 293T or Vero E6 cells implying that the transmembrane domain of 7a is essential for apoptosis induc tion Fig 3A The same mutants were tested in coimmuno precipitation experiments for their abilities to bind Bcl X L The results showed that the 7a 1 116aa mutant Fig 3B lane 3 but not the 7a 1 97aa and 7aH900498 116aa mutants Fig 3B lanes 5 and 7 respectively could bind Bcl X L indicating that the transmembrane domain of 7a is also important for its interaction with Bcl X L In this experiment we observed that the 7a protein has a higher molecular mass form that migrates much more slowly than the monomeric 7a protein Fig 3B top panel lane 1 In order to determine the nature of this higher molecular mass form of 7a a new deletion mutant that lacks the last 4 aa of the transmembrane domain 7a 1 112aa was constructed and an alyzed together with full length 7a and the 7a 1 97aa mutant The Western blot analysis was performed using two sets of lysates one set contained 24 H9262g of total protein Fig 4 lanes 1 to 3 so that the dimeric and or oligomeric forms of 7a are more clearly illustrated while the second set contained 6 H9262gof total protein so that the migrations of the 7a proteins are clearer Fig 4 lanes 4 to 6 As shown in Fig 4 lanes 1 and 2 higher molecular mass forms of full length 7a and the 7a 1 112aa mutant are detected suggesting that they could be dimers and or oligomers which are resistant to boiling 20 mM dithiothreitol and 1 SDS which are found in the Laemmli SDS loading buffer For example the full length 7a protein that migrated at H1101125 kDa Fig 4 lane 1 would match the size of the dimeric form of 7a since the monomeric form of 7a is predicted to be 12 5 kDa Nevertheless the majority of the full length 7a protein migrated at H1101112 5 kDa Fig 4 lanes 1 and 4 suggesting that a greater portion of full length 7a exists as monomers While the 7a 1 112aa deletion mutant showed a more pronounced tendency to form oligomers Fig 4 lane 2 the 7a 1 97aa deletion mutant which lacks the transmem brane domain showed little tendency to form dimers or oli gomers Fig 4 lanes 3 and 6 Overall these results suggested that the higher molecular mass forms of 7a arose from the transient unfolding of the transmembrane helix which resulted in unfavorable exposure of hydrophobic residues and self ag gregation of 7a The deletion of the last 4 amino acids of the transmembrane helix i e the 7a 1 112aa mutant led to fur ther destabilization of the helix and hence more aggregation is observed Amino acids 224 and 225 within the C terminal transmem brane domain of Bcl X L are essential for interacting with the SARS CoV 7a protein In order to determine which domain s in Bcl X L is involved in the interaction with 7a coimmunopre FIG 2 Interactions of the SARS CoV 7a protein with members of the Bcl 2 family A 293T cells were transfected with cDNA constructs expressing full length 7a and myc tagged GST negative control myc GST or myc tagged members of the Bcl 2 family myc Bcl X L myc BAD myc BID myc BAX or myc BAK The cells were harvested at H1101116 h posttransfection lysed and subjected to IP with myc polyclonal antibody and protein A beads The amount of 7a that coimmunopre cipitated with the myc tagged proteins IP anti myc was d