【病毒外文文獻(xiàn)】2006 7a Protein of Severe Acute Respiratory Syndrome Coronavirus Inhibits Cellular Protein Synthesis and Activates p38 M
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JOURNAL OF VIROLOGY Jan 2006 p 785 793 Vol 80 No 2 0022 538X 06 08 00H110010 doi 10 1128 JVI 80 2 785 793 2006 Copyright 2006 American Society for Microbiology All Rights Reserved 7a Protein of Severe Acute Respiratory Syndrome Coronavirus Inhibits Cellular Protein Synthesis and Activates p38 Mitogen Activated Protein Kinase Sarah A Kopecky Bromberg Luis Martinez Sobrido and Peter Palese Department of Microbiology Mount Sinai School of Medicine New York New York 10029 6574 Received 20 July 2005 Accepted 16 October 2005 It was recently shown that the 7a protein of severe acute respiratory syndrome coronavirus induces bio chemical changes associated with apoptosis In this study the mechanism by which the 7a protein induces apoptosis was examined The 7a protein was tested for the ability to inhibit cellular gene expression because several proapoptotic viral proteins with this function have previously been identified 7a protein inhibited expression of luciferase from an mRNA construct that specifically measures translation whereas inhibitors of transcription and nucleocytoplasmic transport did not The inhibition of translation and other cellular processes of gene expression have been associated with the induction of a stress response in cells Western blot analysis using phosphospecific antibodies indicated that 7a protein activated p38 mitogen activated protein kinase MAPK but not c Jun N terminal protein kinase stress activated protein kinase Taken together these data indicate that the induction of apoptosis by the 7a protein may be related to its ability to inhibit cellular translation and activate p38 MAPK Severe acute respiratory syndrome coronavirus SARS CoV has been identified as an enveloped positive sense RNA virus containing a large genome that encodes proteins ex pressed from full length and subgenomic mRNAs The genomic organization of SARS CoV consists of a large repli case gene that is predicted to encode two polyproteins that undergo cotranslational proteolytic processing The replicase gene is followed by several genes encoding structural proteins as well as several predicted nonstructural proteins that are not well characterized and are not encoded by other coronaviruses 21 28 It is possible that one or more of these proteins may contribute to the high pathogenicity caused by SARS CoV SARS CoV spread worldwide in 2003 infecting thousands of people and killing hundreds While it has been demon strated that death was caused by respiratory illness the mo lecular mechanisms of the viral pathogenesis have not been precisely determined Patients infected with SARS CoV de velop severe pneumonia like symptoms but the virus can be found in several other organs such as the kidney and the liver 4 The immune systems of SARS patients are also affected by the disease There is a decrease in CD4 H11001 and CD8 H11001 T cells that begins early in the disease and persists for several weeks 37 The extent of the lymphocyte depletion varies among individuals and a dramatic reduction in the levels of lympho cytes appears to correlate with severe disease symptoms 17 18 As high amounts of virus have been detected in lympho cytes taken from SARS patients 35 it is possible that the lymphocytes are depleted as a direct result of virus induced apoptosis High titers of virus are also found in the lungs suggesting that virus induced apoptosis may also contribute to lung pathology SARS CoV was reported to induce apoptosis in tissue culture cells supporting the hypothesis that virus induced apoptosis may have a role in disease progression 38 Recently the SARS CoV 7a protein also referred to as open reading frame ORF 8 X4 and U122 was demon strated to cause biochemical changes associated with apoptosis in transfected cells 32 The 7a protein has been shown to be expressed in SARS CoV infected tissue culture cells and in lung tissue obtained from SARS CoV patients 3 6 It has also been revealed that 7a protein coimmunoprecipitates with another SARS CoV protein 3a protein also known as ORF 3 ORF 3a X1 and U274 suggesting that the 7a protein and the 3a protein may interact in virus infected cells 33 Comparison of the 7a amino acid sequence with those of other known human and viral proteins yielded no homology The 7a protein is 122 amino acids long has a signal sequence has a predicted transmembrane helix from residues 95 to 117 and is likely a membrane protein 6 The function of 7a protein and its role in the pathogenesis caused by SARS CoV are not well char acterized Part of the 7a protein amino acids 16 to 80 has been crystallized and the structure was resolved 23 The luminal domain of 7a protein adopts a compact immunoglob ulin like H9252 sandwich fold This fold is present in many different proteins including cell surface receptors transcription factors and enzymes and is not indicative of the function of 7a pro tein In the present study it was confirmed that 7a protein in duces apoptosis by analysis of both morphological and bio chemical changes associated with apoptosis In addition it was determined that 7a protein inhibits cellular gene expression Further analysis revealed that 7a protein inhibits cellular gene expression at the level of translation There are discrepancies in the literature as to the localization of 7a protein The data presented here indicate that 7a protein is in fact localized to the Golgi It was also determined that expression of the 7a Corresponding author Mailing address Department of Microbi ology Mount Sinai School of Medicine New York NY 10029 6574 Phone 212 241 7318 Fax 212 534 1684 E mail peter palese mssm edu 785 on March 7 2015 by MAHIDOL UNIV FAC OF MED http jvi asm org Downloaded from protein activates p38 mitogen activated protein kinase MAPK which is also activated in SARS CoV infected cells 22 MATERIALS AND METHODS Cells and plasmids 293T A549 and HeLa cells were cultured in Dulbecco s modified Eagle medium containing 10 fetal bovine serum The enhanced green fluorescent protein GFP ORF from the plasmid pEGFP c1 Clontech was cloned into the pCAGGS plasmid The 7a protein was amplified by reverse transcription PCR from lysates of cells infected with the Urbani strain of SARS CoV and cloned into GFP or hemagglutinin HA pCAGGS The GFP and HA tags are on the C terminal end of 7a protein pGL3 control luciferase expressing plasmid was purchased from Promega In vitro transcribed mRNA and transfections To synthesisze the luciferase mRNA the luciferase gene was cloned from the pGL3 plasmid into the pGEM 4Z plasmid Promega by using the XbaI and HindIII restriction sites Luciferase pGEM 4Z was linearized with BamHI In vitro transcribed mRNA was synthesized with the message machine T7 Ultra kit Ambion The resulting mRNAs contained 5H11032 caps and 3H11032 poly A sequences For transfections cells were seeded onto 24 well plates 6 well plates or 100 mm plates the monolayers were transfected the following day with 200 ng 1 H9262g or 5 H9262g of plasmid or mRNA and 1 H9262l 5 H9262l or 40 H9262l of Lipofectamine 2000 reagent Invitrogen and brought to a total volume of 300 H9262l 1 2 ml or 8 ml with Optimem Invitrogen respectively Microscopy 293T cells were seeded in six well dishes and transfected with GFP pCAGGS or 7a GFP pCAGGS At the indicated times the cells were analyzed using an inverted Zeiss Axiovert 200 microscope Fig 1 293T cells were seeded in 24 well dishes and transfected with the indicated amounts of 7a GFP pCAGGS Fig 2 Cells were analyzed by microscopy at 24 h posttrans fection HeLa cells were seeded in 24 well dishes on coverslips see Fig 6 At 8 h posttransfection cells were fixed with 5 formaldehyde and permeabilized with 1 Triton X 100 Cells were incubated with blocking buffer phosphate buffered FIG 1 Expression of the fusion protein 7a GFP causes morpho logical changes associated with apoptosis 293T cells were transfected with either GFP plasmid or 7a GFP plasmid for 24 or 48 h as indicated in the figure Phase contrast and fluorescence images were obtained by using an inverted Zeiss Axiovert 200 microscope Images are repre sentative of three independent experiments FIG 2 7a GFP activates caspase 3 A A total of 2 H11003 10 5 293T cells were transfected with GFP plasmid or the indicated amounts of 7a GFP plasmid The total amount of DNA transfected was held constant at 200 ng in all samples by the addition of empty vector plasmid Cells were analyzed by microscopy at 24 h posttransfection B A total of 2 H11003 10 5 293T cells were transfected with GFP plasmid or the indicated amounts of 7a GFP plasmid Cells were harvested at 24 h and capsase 3 activity was measured using a DEVD AFC fluoro genic substrate The amount of caspase 3 activated is expressed in arbitrary fluorescence units The data represent the averages H11006 the standard deviation of three experiments C A total of 2 H11003 10 5 293T cells were transfected with either 200 ng GFP plasmid or 7a GFP plasmid for the times indicated Cells were analyzed for caspase 3 activity as described in the legend to panel A The data represent the averages H11006 the standard deviation of three experiments 786 KOPECKY BROMBERG ET AL J VIROL on March 7 2015 by MAHIDOL UNIV FAC OF MED http jvi asm org Downloaded from saline PBS 0 05 Tween 0 5 bovine serum albumin 0 8 glycine for 5 min and then incubated with primary antibody at a dilution of 1 500 for1hatroom temperature Primary antibodies used were mouse anti cytochrome c Pharmin gen rabbit anti protein disulfide isomerase PDI a kind gift from Domenico Tortorella mouse anti HA tag Sigma and mouse anti 7a a kind gift from Ralph Baric Cells were washed three times with blocking buffer and then incubated with donkey anti mouse Alexa Fluor 488 donkey anti rabbit 594 donkey anti mouse 596 or BODIPY TR all from Molecular Probes at a dilution of 1 500 for 1 h Cells were incubated with 4H11032 6 diamidino 2 phenylin dole dihydrochloride DAPI Molecular Probes for 5 min Cells were washed three times and coverslips were mounted on slides with Aqua Polymount Poly sciences Slides were analyzed by confocal microscopy with Zeiss LSM 510 Meta 293T cells were seeded in 24 well dishes transfected with GFP or 7a GFP plasmid in the absence or presence of SB203580 Sigma stained with DAPI and analyzed at 24 h see Fig 8 Caspase 3 activity assay 293T cells were seeded in 24 well dishes and trans fected with the indicated amounts of GFP plasmid or 7a GFP plasmid for the indicated times The total amount of DNA transfected was held constant at 200 ng in all samples with the addition of empty vector plasmid Cells were lysed and analyzed for caspase 3 activity according to the manufacturer s protocol R Roche Lysates were spun down and protein content was determined by Brad ford assay Bio Rad according to the manufacturer s protocol Aliquots of lysates representing equal amounts of protein were analyzed by SDS polyacryl amide gel electrophoresis SDS PAGE using a 10 acrylamide gel The gels were fixed with 25 2 propanol and 10 glacial acetic acid dried and exposed to film Western blot analysis 293T cells were seeded in 100 mm dishes and trans fected with GFP plasmid or 7a GFP plasmid or treated with 1 H9262g ml anisomycin for the indicated times see Fig 7 Cells were harvested lysed in buffer PBS 1 NP 40 0 5 deoxycholate 0 1 SDS and 1 mM EDTA supplemented with protease inhibitor cocktail Complete Roche and spun down to remove nuclei The protein content was determined by Bradford assay Bio Rad Aliquots of lysates representing equal amounts of protein were analyzed by SDS PAGE The proteins were transferred to a nitrocellulose membrane and probed with anti bodies against phospho c Jun N terminal protein kinase phospho JNK stress activated protein kinase total JNK stress activated protein kinase phospho p38 MAPK and total p38 MAPK Cell Signaling Technology After incubation with an anti rabbit peroxidase labeled secondary antibody Amersham blots were analyzed by chemiluminescence Perkin Elmer Cells were transfected with 7a GFP plasmid in the absence or presence of Z VAD R an inhibitor of translation Cyclo and an inhibitor of nucleocytoplasmic trans port Lep B Host gene expression was measured by cotrans fection of a luciferase plasmid and analysis of luciferase activity as shown in Fig 3 Cellular protein synthesis was measured by cotransfection of in vitro transcribed luciferase mRNA and analysis of luciferase activity By using luciferase mRNA trans lation can be measured without the requirement for transcrip tion and nucleocytoplasmic transport Cells were cotransfected either with luciferase plasmid Fig 4A or luciferase mRNA Fig 4B and with 7a GFP plasmid or with GFP plasmid and treated with the inhibitors As in the data shown in Fig 3 7a GFP dramatically inhibited cellular gene expression by nearly 90 Fig 4A The synthetic inhibitors of cellular pro cesses were also effective at inhibiting cellular gene expression The results of the cellular translation analysis differ markedly Cyclo was able to effectively inhibit cellular translation as pre dicted However Act D and Lep B did not inhibit cellular translation and actually caused an increase in cellular transla tion over the GFP samples This is likely due to the fact that the absence of new transcripts caused the mRNAs present to be translated at a higher rate since there was reduced compe tition for translation machinery 7a GFP inhibited cellular translation by nearly 50 suggesting that the mechanism of inhibition of cellular gene expression by 7a protein is transla tion 7a protein also inhibited translation in A549 cells data not shown There was less apparent inhibition of translation by 7a protein Fig 4A than inhibition of total cellular gene expression Fig 4B 50 to 90 This is likely because the luciferase mRNA is expressed rapidly after transfection while it takes several hours for the 7a GFP to be expressed from a plasmid Thus luciferase is probably expressed before the 7a protein inhibition is apparent Indeed it takes 6 h for 7a GFP to be visible in a fluorescence microscope after transfection data not shown There was greater inhibition of translation by 7a protein 74 8 H11006 13 7 when cells were transfected with 7a protein for 12 h and then transfected with luciferase mRNA for an additional 12 h To confirm the inhibition of protein synthesis by 7a protein 293T cells were transfected with a GFP plasmid or a plasmid expressing 7a GFP and pulsed with 35 S methionine at 4 8 or 12 h posttransfection for 10 min Cells were washed harvested and lysed and lysates were analyzed by SDS PAGE The gel was exposed to film and a representative experiment is shown in Fig 5 By this technique only proteins synthesized during a 10 min period at each time point were analyzed There was little difference in translation levels of cells expressing GFP and 7a GFP at 4 h posttransfection Cells transfected with 7a GFP displayed reduced protein synthesis at 8 h and even greater inhibition of protein synthesis at 12 h than cells trans FIG 3 7a GFP inhibits cellular gene expression A total of 2 H11003 10 5 293T cells were transfected with the 200 ng luciferase expressing plas mid pGL3 and either 200 ng GFP plasmid or 200 ng 7a GFP plasmid Cells were harvested at the indicated times and analyzed for luciferase activity by using a luciferase substrate The amount of luciferase present is expressed in arbitrary luminescence units The data repre sent the averages H11006 the standard deviation of three experiments FIG 4 7a GFP inhibits expression of luciferase from an mRNA construct A total of 2 H11003 10 5 293T cells were transfected with either 200 ng pGL3 A or 1 ng luciferase mRNA B and 200 ng GFP plasmid or 200 ng 7a GFP plasmid as indicated Some samples were also treated with Act D Cyclo or Lep B at the time of transfection Cells were harvested at 12 h posttransfection and analyzed for lucif erase as described in the legend to Fig 3 The data represent the averages H11006 the standard deviation of three experiments 788 KOPECKY BROMBERG ET AL J VIROL on March 7 2015 by MAHIDOL UNIV FAC OF MED http jvi asm org Downloaded from fected with GFP These results were confirmed by transfecting a plasmid encoding an HA tagged 7a protein data not shown Thus the data shown in Fig 4 and 5 indicate that 7a protein inhibits protein synthesis The 7a protein localizes to the Golgi apparatus The inhi bition of cellular protein synthesis by 7a protein was unex pected since 7a protein was not predicted to be in the cytosol where the translation machinery is located There are discrep ancies in the literature as to the cellular localization of the 7a protein The 7a protein contains a predicted signal sequence and is thought to be synthesized in the endoplasmic reticulum ER A report indicates that it is retained there 6 but other reports suggest that it is transported to the Golgi 23 To determine the localization of 7a protein GFP and 7a GFP were transfected into HeLa cells and stained with markers for intracellular organelles 293T cells were not used for these experiments because they are quite small and the organelles are difficult to distinguish in the confocal microscope In these experiments the Golgi was visualized using the BODIPY TR Golgi marker the ER was visualized with an antibody to PDI the mitochondria were visualized with an antibody to cyto chrome c and the DNA was visualized with DAPI Represen tative images are shown in Fig 6 Cells expressing GFP Fig 6A displayed GFP fluorescence throughout the nucleus and cytoplasm but GFP did not localize in intracellular compart ments as seen by the absence of yellow overlay in the cells labeled with the Golgi ER and mitochondrial markers In contrast 7a GFP strongly colocalized with the Golgi marker Fig 6B These results were confirmed by using another Golgi marker wheat germ agglutinin data not shown 7a GFP did FIG 5 7a GFP inhibits cellular protein synthesis A total of 10 6 293T cells were transfected with either 1 H9262g GFP plasmid or 1 H9262gof 7a GFP plasmid At 4 8 and 12 h posttransfection cells were labeled with 35 S methionine for 10 min and lysates were analyzed by SDS PAGE Dried gels were exposed to film A representative image from three independent experiments is shown FIG 6 7a GFP is localized to the Golgi A total of 2 H11003 10 5 HeLa cells were transfected with either GFP plasmid A 7a GFP plasmid B 7a HA plasmid C and D bottom row or untagged 7a plasmid D top two rows for 8 h Cells were fixed permeabilized and stained for either the Golgi with BODIPY TR the ER with an antibody to PDI the mitochondria with an antibody to cytochrome c or chromatin with a DAPI stain Untagged 7a protein was visualized with 7a antisera provided by Ralph Baric Green coloring represents GFP A 7a GFP B 7a HA C and untagged 7a D top two rows or 7a HA D bottom row Red coloring represents the indicated organelle and blue coloring represents the chromatin Images are representative of three independent experiments VOL 80 2006 SARS 7a PROTEIN INHIBITS TRANSLATION AND ACTIVATES p38 789 on March 7 2015 by MAHIDOL UNIV FAC OF MED http jvi asm org Downloaded from not appear to localize to the ER mitochondria or nucleus Thus 7a GFP is likely synthesized in the ER and then quickly localizes to the Golgi In addition a 7a construct was tested that contained an HA tag Fig 6C The 7a HA construct also localized to the Golgi but did not localize to the ER mito chondria or nucleus To rule out the possibility that the tags altered localization of 7a protein an untagged 7a protein was also tested Fig 6D top two rows The untagged 7a protein was visualized with 7a antisera which colocalized well with the tagged forms of 7a protein 7a HA Fig 6D bottom row The untagged 7a protein also localized to the Golgi Our re sults agreed with those of Nelson et al where it was described that 7a localizes to the Golgi apparatus in SARS CoV infected cells 23 p38 MAPK but not JNK 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