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JOURNAL OF VIROLOGY Mar 2005 p 3182 3186 Vol 79 No 5 0022 538X 05 08 00H110010 doi 10 1128 JVI 79 5 3182 3186 2005 Copyright 2005 American Society for Microbiology All Rights Reserved Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is a Viral Structural Protein Naoto Ito 1 2 Eric C Mossel 1 Krishna Narayanan 1 Vsevolod L Popov 3 Cheng Huang 1 Taisuke Inoue 1 Clarence J Peters 1 3 and Shinji Makino 1 Departments of Microbiology and Immunology 1 and Pathology 3 The University of Texas Medical Branch at Galveston Galveston Texas and Laboratory of Zoonotic Diseases Division of Veterinary Medicine Faculty of Applied Biological Science Gifu University Gifu Japan 2 Received 4 August 2004 Accepted 20 October 2004 The present study showed the association of a severe acute respiratory syndrome coronavirus SCoV accessory protein 3a with plasma membrane and intracellular SCoV particles in infected cells 3a protein appeared to undergo posttranslational modifications in infected cells and was incorporated into SCoV parti cles establishing that 3a protein was a SCoV structural protein Severe acute respiratory syndrome SARS coronavirus SCoV is the etiological agent of a new emerging infectious disease SARS which originated in southern China in 2002 and spread to various areas of the world in the 2003 epidemic 5 11 12 23 29 Like all coronaviruses the SCoV genome car ries the most 5H11032 end gene 1 which encodes proteins for viral RNA synthesis four viral structural protein genes S M E and N and several about eight putative accessory genes 16 25 28 The biological functions of coronavirus accessory proteins are poorly characterized In most cases they are dispensable for virus replication in cell culture while some appear to con tribute to viral pathogenesis 1 3 21 22 30 31 In mouse hepatitis virus the accessory protein I protein is a viral struc tural protein 9 Among the putative SCoV accessory genes the 3a gene product 3a protein was detected in SCoV in fected cells and in the lungs of SARS patients 32 the 3a gene is also called X1 25 and U274 27 Topology prediction of 3a protein based on the amino acid sequence suggests that 3a is a type III transmembrane protein with three transmembrane domains To examine 3a protein synthesis in SCoV infected cells ac cumulation of 3a protein in Caco2 cells that were infected with the Urbani strain of SCoV was examined by Western blot analysis with anti 3a antibody Anti 3a antibody was prepared by injection of a purified glutathione S transferase GST 3a fusion protein amino acids 127 to 274 of 3a protein were fused with the C terminus of GST protein expressed in Escherichia coli into a rabbit and subsequent affinity purification of the serum by the GST 3a fusion protein Several 3a related signals were detected in SCoV infected cells and not in mock infected cells Fig 1 Among them a 31 kDa protein which corre sponded to the predicted size 30 9 kDa of 3a protein was most abundant Fig 1 arrow Translation of in vitro synthe sized capped RNA transcripts carrying the 3a gene in rabbit reticulocyte lysate also resulted in the production of 31 kDa protein data not shown suggesting that the major 31 kDa protein represented an unmodified 3a protein in infected cells Besides the 31 kDa major band several larger faint bands ranging from 34 to 41 5 kDa were detected suggesting post translational modification of the protein The nature of 3a protein modification requires further studies The presence of two smaller bands of approximately 25 5 and 26 5 kDa in in fected cells suggested that some 3a protein molecules under went specific proteolytic processing and or that these two sig nals represented quasistable degradation products Analysis of the subcellular distribution of 3a protein by con focal microscopy with anti 3a antibody demonstrated the cyto plasmic localization of 3a protein in SCoV infected cells Fig 2A Strong signals were also detected at peripheral regions of the infected cells No signal was detected in mock infected cells Fig 2B Analysis of the subcellular localization of 3a protein in SCoV infected Caco2 cells by immunoelectron microscopy with anti 3a antibody showed that 3a protein was localized in the cytoplasm as well as at the plasma membrane Fig 3A and B whereas the nucleus of the infected cells Fig 3A and uninfected cells one cell at the upper left corner in Fig 3A also Fig 3D showed only background signals More impor tantly 3a protein was distributed in cytoplasmic Fig 3A and C and plasma membrane Fig 3B V regions in which many virus particles had accumulated Essentially the same subcellular localization of 3a protein was detected in infected Vero E6 cells data not shown To eliminate the possibility of nonspecific binding of anti 3a antibody to SCoV we counted the number of intracellular virus particles labeled with 3a specific immunogold signals in immunoelectron micrographs of SCoV infected Caco2 cells stained with anti 3a antibody or the preimmune serum Out of 200 virus particles counted ran domly 75 particles 37 5 had the immunogold label in the cells stained with anti 3a antibody while only 7 3 5 had signals in the cells stained with preimmune serum The differ ence was statistically significant as determined by the chi square test P H11021 0 001 establishing the association of 3a protein with SCoV These data suggested that 3a protein could be incorporated into SCoV particles The data obtained from 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 N I and E C M contributed equally to this study 3182 on March 15 2015 by guest http jvi asm org Downloaded from these microscopic analyses were consistent with the report of 3a protein association with cytoplasmic membrane in 3a pro tein expressing cells 27 and a confocal microscopic study demonstrating that 3a protein was distributed over the cyto plasm and was partly concentrated in the Golgi apparatus of infected cells 32 To test the possibility that 3a protein is a viral structural protein the presence of 3a protein in the purified SCoV was examined To minimize the possible copurification of intracel lular proteins with the purified SCoV we propagated SCoV in Caco2 cells which did not show any visible cytopathic effects during SCoV replication 17 Prior to SCoV purification cul ture supernatants from SCoV infected Caco2 cells were irra diated frozen with 2 H11003 10 6 rads from a Gammacell 60 Co source model 109A JL Shepherd and Associates San Fernando Calif After confirmation of the complete inactivation of virus infectivity the sample was clarified by centrifugation at 450 H11003 g for 15 min SCoV particles were partially purified by two subsequent ultracentrifugations on a discontinuous sucrose gradient consisting of 60 50 30 and 20 sucrose with the use of a Beckman SW28 rotor 10 19 the sample was first cen trifuged at 28 000 rpm for 3 h and the virus particles at the interface of 30 and 50 sucrose were further centrifuged at 28 000 rpm for 18 h The virus particles at the interface of 30 and 50 sucrose were collected diluted and then further applied on a continuous sucrose gradient of 20 to 60 sucrose The samples were centrifuged at 28 000 rpm for 18 h Subse quently 10 fractions were collected and sucrose density in each fraction was measured SCoV in each fraction was pel leted through a 20 sucrose cushion at 38 000 rpm for 2 h with a Beckman SW41 rotor The pellet in each fraction was sub jected to Western blot analysis with anti N protein antibody kindly provided by Xiao Hua Li anti M protein antibody Abgent San Diego Calif and anti 3a antibody Fig 4A Analysis of N protein and M protein demonstrated the distri bution of the purified SCoV in the continuous sucrose gradi ent The strongest signals of N and M proteins were detected in fractions 6 sucrose density 1 185 g ml and 7 sucrose density 1 160 g ml suggesting that the buoyant density of SCoV was similar to that of mouse hepatitis virus 14 15 In addition to N protein of approximately 50 kDa a signal of H1101140 kDa was also detected in the purified SCoV The 40 kDa species of N protein which was barely detected in infected cells was probably produced from the 50 kDa N protein by proteolytic processing N protein of transmissible gastroenter itis coronavirus is cleaved by activated caspases in infected cells 6 while the mechanism of proteolytic processing of SCoV N protein is unknown Multiple M protein signals indicated the presence of various glycosylated forms of M protein in SCoV particles Like N and M proteins several 3a protein related signals were detected in fractions from 5 to 8 with the highest signal in fractions 6 and 7 Although most of the intracellular 3a related signals were detected in purified SCoV a 25 5 kDa signal was not detected in the purified SCoV Also two signals asterisks which were prominent in purified SCoV were not detected in infected cells These data strongly suggested that 3a protein was a viral structural protein To eliminate a possibility that the 3a protein detected in purified SCoV represented a copurified intracellular 3a pro tein contaminant that was released into the culture fluids or that was associated with cell debris Western blot analysis was performed to examine whether the intracellular host protein actin and the SCoV gene 1 protein nsp1 24 which is be lieved to be a nonstructural protein were also found in the FIG 1 Western blot analysis of 3a protein Human colonic adeno carcinoma Caco2 cells were infected with SCoV at a multiplicity of infection of 0 01 and cells were solubilized with sodium dodecyl sul fate polyacrylamide gel electrophoresis sample buffer 100 mM Tris HCl pH 6 8 4 sodium dodecyl sulfate 0 2 bromophenol blue 20 glycerol and 200 mM beta mercaptoethanol at 5 days p i Cell extracts were applied to a sodium dodecyl sulfate 15 polyacrylamide gel and Western blot analysis was performed using anti 3a antibody Lane 1 SCoV infected Caco2 cells lane 2 mock infected Caco2 cells Arrow major 31 kDa 3a protein FIG 2 Confocal microscopic analysis of 3a protein in SCoV in fected cells Vero E6 cells growing in eight well chamber slides Lab Tek Naperville Ill were infected with SCoV at a multiplicity of infection of 1 A or mock infected B At 24 h p i cultures were incubated overnight with 4 paraformaldehyde and then treated with 0 25 Triton X 100 for 15 min Subsequently cells were incubated with anti 3a antibody and goat anti rabbit secondary antibody conju gated with Alexa Fluor 488 dye Molecular Probes Eugene Oreg Cells were observed under the Zeiss LSM 510 UV META laser scan ning confocal microscope in the University of Texas Medical Branch Infectious Disease and Toxicology Optical Imaging Core VOL 79 2005 NOTES 3183 on March 15 2015 by guest http jvi asm org Downloaded from 3184 NOTES J VIROL on March 15 2015 by guest http jvi asm org Downloaded from purified SCoV Fig 4B detection of actin and nsp1 proteins in the purified SCoV sample would indicate that the virus purification procedure was not appropriate Antiactin goat polyclonal immunoglobulin G I 19 Santa Cruz Biotechnol ogy Santa Cruz Calif was used to detect actin Anti nsp1 antibody was raised in rabbits by immunizing them with the synthetic peptide N RKNGNKGAGGHSYG C Analysis of intracellular proteins from uninfected Caco2 cells and infected Caco2 cells at 5 days postinfection p i showed the presence of nsp1 only in infected cell extracts and actin in both cell extracts Both actin and nsp1 proteins were not detected in the purified SCoV Consistent with the data shown in Fig 4A N and 3a proteins were detected in purified SCoV and SCoV infected cells but not in uninfected cells These data strongly indicated that 3a protein in the purified SCoV sample was not a copurified intracellular 3a protein contaminant Our electron microscopic analysis convincingly showed the association of 3a protein with intracellular SCoV as well as SCoV particles at the plasma membrane Fig 3 Western blot analysis demonstrated the presence of 3a protein in the puri fied SCoV Fig 4A The species of 3a protein detected in the purified virion was not identical to that observed in the in fected cells indicating that 3a protein found in the purified SCoV was not an intracellular 3a protein contaminant Fig 4 The absence of actin and SCoV nsp1 proteins in the purified SCoV sample further confirmed that our virus purification method was appropriate Fig 4B Based on these data we concluded that the 3a protein was a viral structural protein A recent study revealed that a highly purified transmissible gas troenteritis coronavirus preparation is possible by the use of an immunopurification method 7 However we were unable to use this new method to purify SCoV due to the lack of an appropriate monoclonal antibody Analysis of highly purified SCoV samples by the immunopurification method may be use ful to study the stoichiometric amount of 3a protein in the purified SCoV relative to other structural proteins Assembly of coronavirus S protein E protein and nucleo capsid is mediated by binding of these molecules to M protein in infected cells 4 8 10 13 18 20 26 Coimmunoprecipita tion studies demonstrated that expressed 3a protein interacts with coexpressed SCoV M E and S proteins 27 A recent study using a crude SCoV preparation indicated the presence of interaction between 3a protein and S protein 33 Although these interactions have not been experimentally demonstrated in infected cells the presence of 3a protein in SCoV indicates that the incorporation of 3a protein into SCoV particles was mediated by the interaction with viral envelope proteins at the virus budding sites Further studies are required to determine whether association of 3a protein with SCoV particles has some role in SCoV assembly 3a protein carries a signal that may be important for rapid internalization of the protein from the plasma membrane 27 If 3a protein interacts with a host protein s in the plasma membrane then there is a possibility that the complex of this putative host protein and 3a protein may be rapidly internal ized It will be interesting to test whether virion associated 3a protein downregulates the expression of some of the mem FIG 4 Western blot analysis of N M and 3a proteins in purified SCoV Caco2 cells were infected with SCoV at a multiplicity of infec tion of 1 and culture fluid was collected at 5 days p i Released SCoV was purified by sucrose gradient centrifugation as described in the text A Ten fractions from a 20 to 60 sucrose gradient containing the virus particles were collected and numbered from bottom B to top T of the gradient The top panel represents the density of each sucrose fraction IC intracellular proteins from SCoV infected Caco2 cells B Purified SCoV lane 1 cell extracts from SCoV infected Caco2 cells at 5 days p i lane 2 and uninfected Caco2 cells at 5 days p i lane 3 were analyzed for actin protein N protein 3a protein and SCoV nsp1 protein FIG 3 Immunogold labeling of 3a protein in SCoV infected cells Caco2 cells were infected with SCoV at a multiplicity of infection of 0 5 fixed at 48 h p i and embedded in LR White resin Ultrathin sections of the cells were incubated with anti 3a antibody and goat anti rabbit immunoglobulin G heavy plus light conjugated to 15 nm colloidal gold particles Amersham Biosciences A In a SCoV infected cell the label is clearly associated with intracellular virus v and with the virions at the cell surface arrows An uninfected cell in the upper left corner is devoid of label n nucleus Bar H11005 1 H9262m B 3a protein is associated with plasma membranes V SCoV particles associated with 3a protein C Association of 3a protein with intracellular virus particles arrows D Portion of cytoplasm of a mock infected Caco2 cell showing occasional staining of a few gold particles near the plasma membrane Bars B to D H11005 0 5 H9262m VOL 79 2005 NOTES 3185 on March 15 2015 by guest http jvi asm org Downloaded from brane associated host proteins to evade host immune re sponses and or alter the cellular environment to one that is suitable for virus replication We thank Bo Xu at the Biomolecular Resource Facility University of Texas Medical Branch for valuable support for preparation of anti 3a antibody and Xiao Hua Li at The Center for Biomedical In ventions University of Texas Southwestern at Dallas for anti N anti body We are also grateful to Eugene P Knutson at the Infectious Disease and Toxicology Optical Imaging Core University of Texas Medical Branch for confocal microscopic analysis and Violet C Han at the Electron Microscopy Laboratory at University of Texas Medical Branch Department of Pathology for expert assistance in electron microscopy This work was supported by Public Health Service grant AI29984 to S M and contract AI25489 to C J P from the National Institutes of Health N I was supported by a fellowship for long term overseas research for young investigators sponsored by the Ministry of Educa tion Culture Sports Science and Technology Japan E C M and C H were supported by NIH fellowship AI007536 and the James W McLaughlin Fellowship Fund respectively REFERENCES 1 Britton P K L Mawditt and K W Page 1991 The cloning and sequenc ing of the virion protein genes from a British isolate of porcine respiratory coronavirus comparison with transmissible gastroenteritis virus genes Virus Res 21 181 198 2 Chen C M D Cavanagh and P Britton 1995 Cloning and sequencing of a 8 4 kb region from the 3H11032 end of a Taiwanese virulent isolate of the coronavirus transmissible gastroenteritis virus Virus Res 38 83 89 3 de Haan C A P S Masters X Shen S Weiss and P J Rottier 2002 The group specific murine coronavirus genes are not essential but their deletion by reverse genetics is attenuating in the natural host Virology 296 177 189 4 de 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