【病毒外文文獻】2006 Severe Acute Respiratory Syndrome Coronavirus 7a Accessory Protein Is a Viral Structural Protein

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JOURNAL OF VIROLOGY Aug 2006 p 7287 7294 Vol 80 No 15 0022 538X 06 08 00H110010 doi 10 1128 JVI 00414 06 Copyright 2006 American Society for Microbiology All Rights Reserved Severe Acute Respiratory Syndrome Coronavirus 7a Accessory Protein Is a Viral Structural Protein Cheng Huang 1 Naoto Ito 1 2 Chien Te K Tseng 1 and Shinji Makino 1 Department of Microbiology and Immunology The University of Texas Medical Branch at Galveston Galveston Texas 77555 1019 1 and Laboratory of Zoonotic Diseases Division of Veterinary Medicine Faculty of Applied Biological Science Gifu University 1 1 Yanagido Gifu 501 1193 Japan 2 Received 27 February 2006 Accepted 16 May 2006 Severe acute respiratory syndrome coronavirus SCoV 7a protein is one of the viral accessory proteins In expressing cells 7a protein exhibits a variety of biological activities including induction of apoptosis activa tion of the mitogen activated protein kinase signaling pathway inhibition of host protein translation and suppression of cell growth progression Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay in which intact SCoV particles were specifically immu noprecipitated by anti S protein monoclonal antibody demonstrated that 7a protein was associated with purified SCoV particles Coexpression of 7a protein with SCoV S M N and E proteins resulted in production of virus like particles VLPs carrying 7a protein while 7a protein was not released from cells expressing 7a protein alone Although interaction between 7a protein and another SCoV accessory protein 3a has been reported 3a protein was dispensable for assembly of 7a protein into VLPs S protein was not required for the 7a protein incorporation into VLPs and yet 7a protein interacted with S protein in coexpressing cells These data established that in addition to 3a protein 7a protein was a SCoV accessory protein identified as a SCoV structural protein Human coronavirus infections typically cause only mild or moderate diseases 19 43 so the revelation that the newly identified severe acute respiratory syndrome SARS corona virus SCoV is the etiologic agent for the global outbreak of life threatening SARS in the winter of 2002 2003 was surpris ing 7 17 18 34 Bats appear to be natural reservoirs of SCoV and SCoV like viruses 20 21 Specific mutations in the viral peplomer protein S protein allowed a SCoV like virus to cross the species barrier and become a highly infectious human pathogen 22 35 SCoV is an enveloped positive sense RNA virus with a spherical shape of approximately 100 nm in diam eter Like other coronaviruses the SCoV membrane contains three viral proteins S M and E The H1101130 kb SCoV genomic RNA is bound with N protein to form a nucleocapsid complex which is surrounded by the viral membrane The genome or ganization of SCoV is also similar to that of other coronavi ruses the 5H11032 two thirds of the genome encodes the gene 1 proteins whose primary functions are associated with viral RNA synthesis and the 3H11032 one third encodes all of the struc tural proteins and accessory proteins including 3a 3b 6 7a 7b 8a 8b and 9b 24 36 40 Expression of 3a 6 and 7a proteins has been confirmed to occur in infected cells and patients 8 10 46 48 SCoV 3a protein is a viral structural protein 15 37 and is released from 3a expressing cells and virus infected cells in membranous structures 13 Although other coronaviruses also produce accessory proteins 19 43 the amino acid sequences of all of the SCoV accessory proteins have no homology with those of any other known viral proteins or nonviral proteins Deletion of open reading frames 3a 3b 6 and 7a either alone or in combination does not affect the virus replication significantly in cell culture 45 demonstrating that these SCoV accessory proteins are not essential for virus rep lication in cell culture Past studies of other coronaviruses suggested that many of the accessory proteins are important for viral virulence in vivo 6 32 33 41 44 Likewise SCoV accessory proteins may play roles in SCoV pathogenesis but the biological functions of these SCoV specific accessory pro teins are largely unexplored The 122 amino acid aa long SCoV 7a protein also known as X4 protein or U122 protein is a type I transmembrane protein consisting of a 15 aa signal peptide sequence at its N terminus an 81 aa luminal domain a 21 aa transmembrane domain and a short C terminal tail 30 The 7a gene is con served in all SCoV strains isolated from humans and animals 21 The crystal structure of the luminal domain of the 7a protein has been resolved revealing an unexpected topology similar to that of members of the immunoglobulin Ig super family 30 There are controversies in the literature as to whether 7a protein is localized in the endoplasmic reticulum or Golgi compartment 8 16 30 Reported biological functions of 7a protein based on expression studies include induction of apoptosis in various cell lines through a caspase dependent pathway 38 inhibition of cellular protein synthesis activation of p38 mitogen activated protein kinase MAPK 16 and suppression of cell cycle progression at the G 0 G 1 phase 47 yet it is still unclear whether 7a protein exerts these functions in infected cells Nevertheless these intriguing results collec tively suggest that 7a protein is likely involved in virus host interactions To further understand the function and properties of SCoV 7a protein we examined whether 7a protein was a SCoV struc Corresponding author Mailing address Department of Microbi ology and Immunology The University of Texas Medical Branch at Galveston Galveston TX 77555 1019 Phone 409 772 2323 Fax 409 772 5065 E mail shmakino utmb edu 7287 on March 18 2015 by ST ANDREWS UNIV http jvi asm org Downloaded from tural protein Our data indicated that in addition to the 3a protein the 7a protein was a viral structural protein We have also established a SCoV virus like particle VLP producing system by expressing SCoV proteins without using exogenous viruses and confirmed that the 7a protein was assembled into SCoV VLPs MATERIALS AND METHODS Cells and virus Human embryonic kidney 293T cells were maintained in Dulbecco s modified Eagle s essential medium supplemented with 10 fetal bovine serum L glutamine 2 mM nonessential amino acids 0 1 mM and kanamycin 100 H9262g ml Human colon carcinoma epithelium Caco2 cells were maintained in minimum essential medium Eagle with 20 fetal bovine serum L glutamine 2 mM nonessential amino acids 0 1 mM and kanamycin 100 H9262g ml Cells were incubated at 37 C in 5 CO 2 The Urbani strain of SCoV was propagated in a biosafety level 3 laboratory as described previously 15 27 Construction of plasmids and transient protein expression Total intracellular RNA was extracted from SCoV infected Vero E6 cells with TRIzol reagent Invitrogen according to the manufacturer s protocol SCoV S M N E 3a and 7a genes were amplified from total RNA prepared from SCoV infected cells by reverse transcription PCR The PCR products were cloned into a mam malian expression vector pCAGGS resulting in pCAGGS S pCAGGS M pCAGGS N pCAGGS E pCAGGS 3a and pCAGGS 7a respectively All of the plasmids were confirmed using sequence analysis The 7a gene was also cloned into pcDNA myc plasmid Invitrogen to generate pcDNA 7a myc in which a myc tag was attached to the C terminus of the 7a protein Cultures of 293T cells grown on 100 mm dishes were transfected with expression plasmids as indicated in each experiment by using TransIT 293 reagent Mirus Madison WI according to the manufacturer s instructions At 48 h posttransfection culture media were collected by centrifugation at 1 500 H11003 g for 10 min at 4 C The media were further clarified by filtration through 0 45 H9262m syringe filters and partially purified by centrifugation through 20 sucrose cushions at 26 000 rpm for3hinaBeckman SW28 rotor Samples were resuspended in 1H11003 sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE loading buffer 62 5 mM Tris HCl pH 6 8 2 SDS 10 glycerol 50 mM dithiothreitol Cells were washed once with ice cold phosphate buffered saline PBS Total cell lysates were prepared by adding 1H11003 SDS PAGE loading buffer All of the samples were incubated at 37 C for 30 min and subjected to Western blot analysis Antibodies and Western blot analysis Production of rabbit anti SCoV 7a protein antibody and rabbit anti SCoV 3a protein antibody was reported previ ously 13 15 Rabbit anti SCoV S polyclonal antibody IGM 541 was obtained from IMGENEX San Diego CA Rabbit anti SCoV M antibody AP6008b was purchased from Abgent San Diego CA Mouse anti SCoV N antiserum was provided by Xiao Hua Li at The University of Texas Southwestern Medical Center at Dallas Mouse anti SCoV S protein monoclonal antibody NR 617 was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository NIAID Horseradish peroxidase HRP conjugated goat anti rabbit IgG HRP conjugated goat anti mouse IgG and HRP conjugated donkey anti goat IgG were purchased from Santa Cruz Biotechnology CA Protein samples were separated by SDS PAGE and transferred onto polyvinyli dene difluoride membranes Bio Rad with a Trans Blot SD semidry transfer apparatus Bio Rad The membranes were blotted with primary antibodies diluted 1 1 000 in PBS containing 5 nonfat milk overnight at 4 C Membranes were washed and incubated with secondary antibodies diluted 1 3 000 for1hat room temperature The protein bands were visualized with ECL reagent Amer sham Biosciences by following the manufacturer s instructions Ultracentrifugation of SCoV samples on continuous sucrose gradients Su pernatants from SCoV infected Caco2 cells were inactivated by irradiation with 2 H11003 10 6 rads from a Gammacell 60 Co source model 109A J L Shepherd and Associates San Fernando CA as described previously 15 Inactivation of virus infectivity was confirmed by tissue culture assay After centrifugation at 1 500 H11003g for 10 min at 4 C samples were further clarified by filtration through 0 45 H9262m syringe filters The clarified samples were partially purified by centrifugation through 20 sucrose cushions at 26 000 rpm for3hinaBeckman SW28 rotor and resuspended in NTE buffer 100 mM NaCl 10 mM Tris HCl pH 7 5 1 mM EDTA Then the samples were applied onto a 20 to 60 continuous sucrose gradient and subjected to centrifugation at 26 000 rpm for 18 h in an SW28 rotor Twelve fractions were collected from the bottom of the gradient and diluted in NTE buffer The SCoV particles in the fractions were pelleted through a 20 sucrose cushion at 38 000 rpm for 2 h using a Beckman SW41 rotor The pellets were dissolved in 1H11003 SDS PAGE loading buffer and used for Western blot analysis Generation and analysis of SCoV VLPs Mixtures of plasmids containing pCAGGS S pCAGGS M pCAGGS N pCAGGS E pCAGGS 3a or pCAGGS 7a in various combinations as indicated in each experiment were transfected into subconfluent 293T cells grown on 100 mm cell culture dishes Culture media were harvested and analyzed using Western blotting as described above In metabolic label experiments the transfected cells were labeled with 100 H9262Ci ml Trans 35 S Label MP Biomedicals CA from 42 h to 48 h after transfection The culture media were collected clarified as described above and centrifuged at 26 000 rpm for3honadiscontinuous sucrose gradient consisting of 60 50 30 and 20 sucrose by using an SW28 rotor The VLP samples at the interface of 30 and 50 sucrose were collected diluted with NTE buffer and further loaded on the top of a 20 to 60 continuous sucrose gradient After 18 h of centrifu gation at 26 000 rpm in an SW28 rotor 10 fractions were collected from the bottom of the gradient and measured for sucrose densities Samples were diluted and recovered by centrifugation through a 20 sucrose cushion in a Beckman SW41 rotor The pellets were dissolved in 1H11003 SDS PAGE loading buffer applied to a 10 SDS PAGE gel and analyzed using autoradiography SCoV virus capture assay A SCoV virus capture assay was performed as reported previously 13 Briefly supernatants from SCoV infected Caco2 cells or transfected cells were partially purified by centrifugation through a 20 sucrose cushion The pellet was suspended in NTE buffer containing a protease inhibitor cocktail Sigma with bovine serum albumin BSA at a concentration of 0 3 Samples were precleared by incubation with 40 H9262l of protein A G Sepharose 4 Fast Flow beads Amersham Biosciences at 4 C for 1 h Then samples were collected and mixed with 5 H9262g of mouse anti SCoV S protein monoclonal antibody NR 617 or mouse anti H2K k D k H2K monoclonal an tibody 29 After incubation at 4 C overnight 5 H9262g of goat anti mouse IgG FcH9253 fragment specific Jackson ImmunoResearch Laboratories Inc West Grove PA was added and incubated at 4 C for 3 h Then 40 H9262l of protein A G Sepharose Fast Flow beads was added and incubated for another 3 h at 4 C After centrifugation at 750 H11003 g for 5 min the supernatant was collected The pellets were washed with NTE buffer five times Finally the collected superna tant and the pellets were suspended in SDS PAGE loading buffer and used for SDS PAGE and Western blot analysis Coimmunoprecipitation analysis Subconfluent 293T cells were transfected with plasmids by means of TransIT 293 reagent At 24 h posttransfection cells were harvested and washed once with ice cold PBS Cell lysates were prepared in radioimmunoprecipitation assay lysis buffer 1 NP 40 0 1 SDS 0 5 sodium deoxycholate and protease inhibitor cocktail in PBS on ice for 30 min and briefly centrifuged using an Eppendorf microcentrifuge at maximum speed to remove cell debris Supernatants were collected and precleared by incubation with 40 H9262l of protein A G Sepharose Fast Flow beads for1hat4 C Then 1 H9262g of rabbit anti 7a protein antibody mouse anti myc monoclonal antibody 9E10 Santa Cruz or mouse anti SCoV S protein monoclonal antibody NR 617 was added as indicated in each experiment to precipitate the 7a protein 7a myc protein or S protein at 4 C overnight The samples were incubated further with 40 H9262l of protein A G Sepharose Fast Flow beads for an additional3hat4 C After centrifugation at 750 H11003 g for 5 min precipitated immune complexes were washed three times with lysis buffer and twice with PBS Finally the immuno precipitated proteins were dissolved in 2H11003 SDS PAGE loading buffer and ana lyzed via Western blot assay RESULTS SCoV 7a protein is a SCoV structural protein To test whether SCoV 7a protein was a viral structural protein SCoV that was released from infected Caco2 cells was purified using a 20 to 60 sucrose gradient centrifugation Twelve fractions were collected from the bottom of the gradient and the pres ence of SCoV proteins in each fraction was analyzed using Western blot analysis Consistent with our past studies 13 the strongest S N and M protein signals were detected in fractions 5 density 1 20 g ml and 6 density 1 18 g ml Fig 1 showing that the buoyant density of SCoV was between 1 18 and 1 20 g ml Western blot analysis clearly demonstrated that the peak of SCoV 7a protein was also in fractions 5 and 6 suggesting that the 7a protein was a SCoV structural protein 7288 HUANG ET AL J VIROL on March 18 2015 by ST ANDREWS UNIV http jvi asm org Downloaded from To further confirm the presence of 7a protein in SCoV particles a virus capture assay using anti SCoV S protein monoclonal antibody was conducted This method allows pu rification of intact SCoV from the culture media of virus infected cells with high specificity and efficacy 13 31 Partially purified SCoV was incubated with anti SCoV S monoclonal antibody and subsequently captured using immunoprecipita tion Our results demonstrated that anti SCoV S monoclonal antibody successfully precipitated intact SCoV particles carry ing M S and 3a proteins Fig 2 SCoV 7a protein was also precipitated by anti SCoV S monoclonal antibody In contrast an unrelated H2K monoclonal antibody failed to precipitate any of these viral proteins exhibiting the specificity of the virus capture assay The virus capture assay was quite effective for precipitating intact SCoV particles as neither the 7a protein nor the M protein the most abundant viral structural protein was detected in the supernatant of the immunoprecipitated samples data not shown Because 7a protein was identified in the SCoV samples purified with two different purification methods we concluded that 7a protein was a novel SCoV structural protein SCoV 3a protein is dispensable for incorporation of 7a pro tein into virions A past study reporting the interaction be tween expressed 3a and 7a proteins 39 led us to examine the possibility that SCoV 7a protein was incorporated into SCoV particles through an interaction between proteins 3a and 7a To this end we have established a SCoV VLP system in which SCoV VLPs were produced from the cells coexpressing viral structural proteins SCoV genes were cloned downstream of the chicken H9252 actin promoter in the pCAGGS mammalian expression plasmid for the expression of viral proteins Sub confluent 293T cells were cotransfected with pCAGGS S ex pressing SCoV S protein pCAGGS M expressing SCoV M protein pCAGGS N expressing SCoV N protein and pCAGGS E expressing SCoV E protein All of these proteins are common coronavirus structural proteins Cells were meta bolically labeled with Tran 35 S for 6 h from 42 to 48 h post transfection At 48 h posttransfection culture media were col lected and clarified Released VLPs were partially purified through ultracentrifugation on a discontinuous sucrose gradi ent for 3 h and subsequently on a continuous sucrose gradient overnight Ten fractions were collected from the bottom and the VLPs in each fraction were pelleted through 20 sucrose cushions Samples were examined using SDS PAGE and au toradiography SCoV VLPs containing S M and N proteins were detected and most of them were found in fraction 6 sucrose density 1 167 g ml Fig 3 We were unable to detect the E protein by separating SCoV VLP proteins in a higher concentration of a gel suggesting a low abundance of E pro tein in SCoV VLPs The role of 3a protein in the assembly of 7a protein into SCoV particles was studied by examining incorporation of 7a protein into VLPs in the presence and absence of SCoV 3a protein Subconfluent 293T cells were transfected with a mix ture of pCAGGS S pCAGGS M pCAGGS N pCAGGS E FIG 1 Association of SCoV 7a protein with purified SCoV parti cles Supernatant from SCoV infected Caco2 cells was clarified and SCoV particles were pelleted down through a 20 sucrose cushion The pellet was suspended in NTE buffer and purified by ultracentrif ugation on a continuous 20 to 60 sucrose gradient Twelve fractions were taken from the bottom The purified viruses in each fraction were pelleted by centrifugation through a 20 sucrose cushion Samples were dissolved in 1H11003 SDS PAGE loading buffer Western blot analysis using anti 7a antibody 7a anti SCoV S protein antibody S anti SCoV N protein antibody N and anti SCoV M protein antibody M was performed to detect virion 7a S N and M proteins respec tively FIG 2 SCoV capture assay SCoV particles in clarified superna tants from SCoV infected Caco2 cells were pelleted down by centrif ugation on a 20 sucrose cushion The suspended pellets were immu noprecipitated by a nonspecific mouse anti H2K monoclonal antibody H9251 H2K or by a mouse anti SCoV S monoclonal antibody H9251 S to capture virions Immunoprecipitated virions were separated by SDS PAGE and subsequently analyzed by Western blotting using rabbit anti SCoV 7a antibody 7a rabbit anti SCoV M antibody M rabbit anti SCoV S protein S and rabbit anti 3a protein 3a to detect viral proteins in the captured virions MH11001IgG L represents a mixture of a minute IgG light chain signal see left lane and a fast migrating major M protein signal VOL 80 2006 SCoV 7a PROTEIN 7289 on March