【病毒外文文献】2007 The SARS-Coronavirus Membrane protein induces apoptosis through modulating the Akt survival pathway

Abstract A number of viral gene products are capable of triggering apoptotic cell death through interfering with cellular signaling cascades including the Akt kinase pathway In this study the pro apoptotic role of the SARS CoV Membrane M structural protein is described We found that the SARS CoV M protein induced apoptosis in both HEK293T cells and transgenic Drosophila We further showed that M protein induced apoptosis involved mitochondrial release of cytochrome c protein and could be suppressed by caspase inhibitors Over expression of M caused a dominant rough eye phenotype in adult Drosophila By performing a forward genetic modiWer screen we identiWed phosphoinositide dependent kinase 1 PDK 1 as a dominant suppressor of M induced apoptotic cell death Both PDK 1 and Akt kinases play essential roles in the cell survival signaling pathway Altogether our data show that SARS CoV M protein induces apoptosis through the modulation of the cellular Akt pro survival pathway and mitochondrial cytochrome c release 2007 Elsevier Inc All rights reserved Keywords Cytochrome c Drosophila HEK293T PDK 1 Severe Acute Respiratory Syndrome In 2003 the Severe Acute Respiratory Syndrome Coro navirus SARS CoV emerged and caused an outbreak of atypical pneumonia worldwide The SARS CoV genome contains 13 15 open reading frames ORFs 1 3 which encode the ORF1a 1b replicases and protease enzymes Spike S Envelope E Membrane M Nucleocapsid N and a number of less well characterized regulatory proteins 1 2 Previous studies revealed that SARS CoV infection triggers a number of cellular responses in infected cells including modulation of signal transduction cascades such as the p38 MAPK and Akt cell survival pathways 4 12 and induction of apoptosis 11 13 14 Apoptosis is a main pathologic feature induced by SARS CoV infection cytopathic eVect CPE 1 in the infected cells 13 but appears not to be a mechanism for dissemination of new virions 15 Over expression of individual viral proteins is now known to be capable of interfering with various cell signaling cascades For example the N protein can modu late the MAPK pathway and down regulate 14 3 3 protein level 12 23 It is proposed that alteration of the status of various cellular signaling cascades such as the Akt pathway through the concerted eVort of individual viral 1 Abbreviations used AO acridine orange CPE cytopathic eVect DIAP1 Drosophila inhibitor of apoptosis 1 E Envelope EGFP en Archives of Biochemistry and Bioph The SARS Coronavirus Membrane through modulating the Ak Chak Ming Chan a b Cheuk Wing Ma a c Wood Y a Laboratory of Drosophila Research The Chinese Univers b Department of Biochemistry The Chinese University of H c Molecular Biotechnology Programme The Chinese Univer d Department of Anatomy The Chinese University of Received 11 October 2006 and in Available online 31 Jan 0003 9861 see front matter 2007 Elsevier Inc All rights reserved doi 10 1016 j abb 2007 01 012 4 11 13 15 and a number of SARS CoV proteins have been shown to be pro apoptotic 12 16 22 Apoptosis induction has further been demonstrated to initiate viral Corresponding author Fax 852 2603 7732 E mail address hyechan cuhk edu hk H Y E Chan ysics 459 2007 197 207 protein induces apoptosis t survival pathway ee Chan d Ho Yin Edwin Chan a b c ity of Hong Kong Shatin N T Hong Kong SAR China ong Kong Shatin N T Hong Kong SAR China sity of Hong Kong Shatin N T Hong Kong SAR China Hong Kong Shatin N T Hong Kong SAR China revised form 20 December 2006 uary 2007 hanced green Xuorescent protein GAPDH glyceraldehyde 3 phosphate dehydrogenase GSK3afii9826 glycogen synthase kinase3afii9826 IAP inhibitor of apoptosis M Membrane MAPK mitogen activated protein kinase N Nucleocapsid ORF opening reading frame PDK 1 phosphoinositide dependent kinase 1 PKCafii9833 protein kinase Cafii9833 S Spike SARS CoV Severe Acute Respiratory Syndrome Coronavirus Ser serine Thr threo nine 198 C M Chan et al Archives of Biochemi proteins would eventually deliver detrimental consequences to SARS CoV infected cells 24 The SARS CoV Membrane locus is located in the 3H11032 region of the viral genome which encodes a protein of 221 amino acids 1 2 The SARS CoV M protein is one of the major proteins among all viral gene products 25 The M protein possesses a triple membrane spanning region an extracellular N terminal and a long cytosolic C terminal domains 26 Physical interactions between M and other viral proteins including N 27 29 S 30 3a 31 and 7a 32 have been elucidated and such protein protein inter action events are predicted to be essential for the biological functions of M in the viral life cycle Postulated roles of M include promoting membrane fusion regulating viral repli cation and packing genomic RNA into viral particles 26 33 Apart from the well documented roles in viral infec tion and propagation novel functions of various SARS CoV structural proteins such as N and S have recently been assigned 12 19 20 22 In the present study we aimed at investigating previously unidentiWed roles of the SARS CoV M protein We over expressed the SARS CoV M pro tein in cells and transgenic Drosophila and observed that M was capable of inducing a mitochondrial mediated caspase dependent apoptosis We also showed that M over expres sion down regulated Akt protein phosphorylation Materials and methods Construction of mammalian expression vector Full length ORF of the SARS CoV Membrane locus was PCR ampli Wed from viral cDNA template CUHK Su10 SARS CoV isolate Gen Bank Accession No AY282752 and subcloned into pcDNA3 1 vector using EcoRI and XbaI enzymes to generate the pcDNA3 1 Membrane construct Mammalian cell culture and transient transfection Human embryonic kidney cell line HEK293T was maintained at 37 C in Dulbecco s modiWed Eagle s medium DMEM Invitrogen supple mented with 10 heat inactivated fetal bovine serum Gibco BRL strep tomycin 100 g ml and penicillin 100 U ml Cells were seeded onto 6 or 24 well plates 24 h prior to transfection Four 6 well plate or 0 8 24 well plate afii9839g of plasmid DNA were used for transient transfection with Lipofectamine 2000 reagent Invitrogen Cells were collected for the subsequent analyses 48 h post transfection Semi quantitative RT PCR analysis Total RNA was prepared as previously described 34 Primers used were GAPDH F 5H11032 ACC ACA GTC CAT GCC ATC AC 3H11032 GAPDH R 5H11032 TCC ACC ACC CTG TTG CTG TA 3H11032 M F 5H11032 ATT ACC GTT GAG GAG CTT AAA CAA 3H11032 and M R 5H11032 CAA TGA CAA GTT CAC TTT CC 3H11032 ImmunoXuorescence staining of HEK293T cells Cells were seeded onto poly D lysine coated coverslips at a density of 2 10 4 cells coverslip After transfection cells were Wxed with 3 7 form aldehyde for 15 min and then permeabilized by 1 Triton X 100 for 5 min After blocking with 1 goat serum for 30 min cells were incubated with rabbit anti SARS virus PUPM antibody C term 1 100 Abgent alone stry and Biophysics 459 2007 197 207 or in combination with mouse anti native cytochrome c clone 6H2 B2 1 100 Pharmingen at 4 C overnight and followed by secondary anti body incubation with goat anti rabbit IgG H L FITC 1 250 Zymed alone or in combination with goat anti mouse IgG H L TRITC 1 250 Zymed at room temperature for 1 h Golgi body was stained by BODIPY TR ceramide complexed to BSA 5 afii9839M Molecular Probes and cell nuclei were labeled with Hoechst 33342 trihydrochloride trihydrate 5 afii9839M Molecular Probes at room temperature for 10 min Cell permeable synthetic caspase inhibitors Merck Z DQMD fmk caspase 3 inhibitor V z IETD fmk caspase 8 inhibitor II and z LEHD fmk caspase 9 inhibitor I were dissolved in DMSO HEK293 cells were treated with caspase inhibitors 50 afii9839M in 1 DMSO 24 h post transfection and were further incubated for another 24 h Fluorescence images were captured using an Olympus BX51 upright Xuorescence microscope or a Leica NT confocal microscope Drosophila genetics Fly strains were grown at 29 C on standard cornmeal medium supple mented with dry yeast PDK 1 1 and PDK 1 2 lines were kind gifts of Jongkyeong Chung 35 gmr GAL4 UAS DIAP1 UAS P35 P GMR Akt1 Exel 2 Exelixis and DrosDel lines were obtained from Blooming ton Drosophila Stock Center and the dc3 EP2305 and PDK 1 EP837 lines were obtained from Szeged Drosophila Stock Centre Generation of transgenic Xy lines Full length ORF of the SARS CoV Membrane gene was PCR ampli Wed from viral cDNA template and subcloned into pUAST vector 36 using EcoRI and XbaI enzymes to generate pUAST Membrane plasmid Standard microinjection technique was employed to generate transgenic lines A total of 10 UAS Membrane transgenic Xy lines were generated When crossed to gmr GAL4 all lines showed dominant rough eye pheno type A transgenic Xy line J3 carrying an UAS Membrane insert on the third chromosome was used in this study Scanning electron microscopy of adult Xy eyes In brief Xy heads were Wxed in 2 5 glutaraldehyde EM grade Electron Microscopy Sciences in phosphate buVer pH 7 4 for 4 h then post Wxed with 1 osmium tetroxide Electron Microscopy Sciences dehydrated to 100 ethanol and critical point dried with liquid CO 2 Gold palladium coated specimens were examined with a Jeol JSM 6301FE microscope operated at 5 kV 37 Acridine orange and immunoXuorescence staining of larval eye discs Acridine orange staining of third instar larval eye discs was performed as previously described 38 QuantiWcation of acridine orange positive cell was performed by Image Pro Plus 5 1 Media Cybernetics Immuno Xuorescence staining was performed as previously described 16 Rabbit anti SARS virus PUPM antibody C term 1 100 Abgent and goat anti rabbit IgG H L FITC secondary antibody 1 250 Zymed were used Propidium iodide 10 afii9839g ml Molecular Probes was used to label cell nuclei Images were captured using an Olympus BX51 upright Xuorescence microscope or a Leica NT confocal microscope Western blot analysis For Xy protein sample preparation 16 adult Xy heads of appropriate genotypes were homogenized in 75 afii9839l of 6 SDS sample buVer SDS PAGE separation and Western blotting were performed as previously described 16 Primary antibodies used include phospho Drosophila Akt Ser505 antibody 1 1000 Cell Signaling total Akt antibody 1 1000 Cell Signaling and anti afii9826 tubulin E7 1 2000 Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by The University of Iowa Department of Biological Sciences Iowa City IA Fig 1 Expression and subcellular localization of SARS CoV Membrane prot was examined by RT PCR Cells transfected with pcDNA3 1 Membrane expression was detected in the untransfected and empty vector controls GAP and absence of reverse transcriptase enzyme in the RT reaction respectively rescence staining The M protein displayed a punctate cytoplasmic staining pa Untransfected cells showed no expression of Membrane protein B Scale bars ein in HEK293T cells A mRNA expression level of the SARS CoV M gene plasmid showed M mRNA expression at 48 h post transfection while no DH was used as loading control RT and RT represent the presence B G Subcellular localization of M protein was determined by immunoXuo C M Chan et al Archives of Biochemistry and Biophysics 459 2007 197 207 199 52242 USA Secondary antibodies used were goat anti rabbit IgG HRP antibody 1 2000 Cell Signaling and goat anti mouse IgG HRP antibody 1 2000 Zymed Statistical analyses Statistical analyses were performed using Student s t test Data were presented as means SEM p values 0 05 were considered statistically signiWcant Results Expression and subcellular localization of the SARS CoV Membrane protein in HEK293T cells To study the function of the SARS CoV Membrane pro tein the M ORF of the CUHK Su10 SARS CoV isolate was subcloned into a mammalian expression vector pcDNA3 1 By RT PCR mRNA expression of the M gene was detected 48 h post transfection Fig 1A Immu noXuorescence was performed to determine the subcellular localization of the M protein We observed that M protein displayed a punctate localization in the cytoplasm Fig 1E and was partially co localized with the Golgi body Fig 1G inset Induction of apoptosis by SARS CoV Membrane protein in HEK293T cells When the cell nuclei of M transfected HEK293 cells were stained with Hoechst dye we found that 56 of the cell nuclei were condensed as indicated by size reduction Fig 2I and M whereas only around 10 of the control cells had condensed nuclei Fig 2B E and M Since nuclear condensation is a hallmark feature of apoptosis we reasoned that M over expression induced apoptosis in HEK293T cells To conWrm this we treated untransfected HEK293T cells with staurosporine a commonly used apoptosis inducer and nuclear condensation was also observed Fig 2K and M Since cytochrome c release from the mitochondria repre sents one form of apoptotic cell death we performed cyto chrome c immunoXuorescence staining to examine whether ttern E in green and partially localized G with the Golgi body F in red represent 16 afii9839m counted in each experiment Scale bars represent 16 afii9839m p 0 05 For interp to the web version of this paper 200 C M Chan et al Archives of Biochemistry and Biophysics 459 2007 197 207 Fig 2 Induction of apoptosis by over expression of M protein in HEK293T cells A L M protein expression induced nuclear condensation in HEK293T cells Expression of M protein was only detected in transfected cells G in green but not in untransfected A and J and empty vector D controls Hoe chst 33342 was used to label cell nuclei B E H and K in blue Cells expressed with M protein showed nuclear condensation I arrowheads at 48 h post transfection Untransfected cells treated with 1 afii9839M staurosporine for 8 h also displayed nuclear condensation K arrowheads while untreated B and empty vector E controls showed normal nuclear morphology M The percentage of cells showed nuclear condensation was quantiWed Results were plotted as percentage of cells showed nuclear condensation and expressed as means SEM of three independent experiments At least 100 cells were retation of the references to color in this Wgure legend the reader is referred Fig 3 Cytochrome c and caspases are involved in M induced apoptosis A F HEK293T cells When cells were either transfected with M C for 48 h or tr protein was observed Untransfected cells were used as control A Respective G M induced nuclear condensation was inhibited by caspase inhibitors at 5 itor II and z LEHD fmk caspase 9 inhibitor I Results were plotted as perc of three independent experiments At least 100 cells were counted in each expe M protein expression induced mis localization of cytochrome c protein in eated with 1 afii9839M staurosporine for 8 h E mis localization of cytochrome c phase contrast images are shown in B D and F Scale bars represent 16 afii9839m 0 afii9839M z DQMD fmk caspase 3 inhibitor V z IETD fmk caspase 8 inhib C M Chan et al Archives of Biochemistry and Biophysics 459 2007 197 207 201 mitochondrial release of cytochrome c was detected in M transfected cells Unlike the untransfected control Fig 3A we found that cells either transfected with M Fig 3C or treated with staurosporine Fig 3E showed extensive mis localization of cytochrome c protein which is indicative of mitochondrial release of cytochrome c protein entage of cells showed nuclear condensation and expressed as means SEM riment p 0 05 localization Fig 4E Fig 4 Induction of rough eye phenotype by over expression of M protein in rough eye phenotype as characterized by loss of regularity of the adult exter over expression of the EGFP protein B showed no dominant external eye phe instar imaginal eye disc tissues The M protein showed a distinct punctate cyto trol protein showed homogeneous intracellular staining E in green Cell nuc erences to color in this Wgure legend the reader is referred to the web version of could suppress M induced apoptosis in Drosophila Both Drosophila A C Over expression of the M protein in eye tissues resulted in nal eye structure C whereas the gmr GAL4 driver alone control A and notype D F Subcellular localization of M protein in the Drosophila third plasmic expression pattern F in green whereas the expression EGFP con 202 C M Chan et al Archives of Biochemistry and Biophysics 459 2007 197 207 39 The appearance of M transfected Fig 3D and staurosporine treated cells Fig 3F were also morphologi cally distinct from the untransfected control Fig 3B We further showed that M induced nuclear condensation in HEK293T cells could be suppressed by caspase 3 8 and 9 inhibitors Fig 3G Over expression of SARS CoV Membrane protein in Drosophila We previously used a transgenic Drosophila model 16 to demonstrate the pro apoptotic role of the SARS CoV 3a protein 17 In this study we established UAS M transgenic Xy lines and over expressed the SARS CoV M protein in the Drosophila eye using GAL4 UAS transgene expression system 36 Over expression of M caused a rough eye phenotype in adult Xies as shown by the loss of regularity of external eye morphology Fig 4C while the over expression of the EGFP control protein Fig 4B did not show any obvious dominant eye malformation pheno type To determine the subcellular localization of M in Drosophila we performed immunoXuorescence staining in third instar larval eye discs Fig 4D and F Similar to mammalian cells Fig 1E M displayed a punctate cyto plasmic localization in Xy cells Fig 4F whereas the EGFP control protein showed homogeneous intracellular Over expression of SARS CoV Membrane protein induced apoptosis in Drosophila Since over expression of many genes would result in rough eye phenotype in Drosophila we therefore per formed standard acridine orange AO staining 38 in third instar larval imaginal eye discs to examine the pro apopto tic roles of M in vivo Acridine orange is a dye that speciW cally stains apoptotic cells and we found that the number of AO positive cells in M over expressing eye discs Fig 5G and K was signiWcantly higher than that of the control Fig 5F and K To further conWrm the pro apop totic role of M we co expressed two anti apoptotic factors P35 a viral caspase inhibitor and the Drosophila inhibitor of apoptosis 1 DIAP1 protein independently with M As expected both the rough eye phenotype Fig 5B D and elevated number of AO positive cells Fig 5G I and K were mostly suppressed back to the control level respec tively We next questioned whether M induced apoptosis would act through the route of cytochrome c in Xies as in HEK293T cells Fig 3C It has been reported that over expression of the apo form of cytochrome c without heme group can block apoptosis 40 We made use of an EP insert line dc3 EP2305 to over express the endogenous apo cytochrome c gene dc3 16 and asked if dc3 over expression lei were stained by propidium iodide in red For interpretation of the ref this paper C M Chan et al Archives of Biochemi sophila in order to gain further insights into the mechanistic details of M action in vivo By analyzing the modifying stry and Biophysics 459 2007 197 207 203 the rough eye phenotype Fig 5B and elevated number of AO positive apoptotic cells in eye discs Fig 5G and K were largely suppressed upon co expression of M with apo cytochrome c Fig 5 E J and K Expression of phosphoinositide dependent kinase 1 dominantly suppressed SARS CoV Membrane induced apoptosis We performed a forward genetic modiWer screen in Dro eVects of overlapping genomic deletion lines from both the Exelixis 41 and DrosDel 42 collections on the M induced rough eye phenotype Fig 4C 30 modifying genomic regions were identiWed C M Chan W M Chan C S Chan C W Ma K W Ching L L Ho K M Lau H Y Chan unpublished observations Detailed characterization of one of these modifying regions 61B1 C1 is described here The 61B1 C1 genomic region is uncovered by a dele tion line Df 3L ED201 42 We further found that one of the EP insert lines in the 61B1 C1 region PDK 1 EP837 dom Fig 5 Induction of apoptosis by over expression of M