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JOURNAL OF VIROLOGY May 2007 p 5423 5426 Vol 81 No 10 0022 538X 07 08 00H110010 doi 10 1128 JVI 02307 06 Copyright 2007 American Society for Microbiology All Rights Reserved Severe Acute Respiratory Syndrome Coronavirus Accessory Protein 6 Is a Virion Associated Protein and Is Released from 6 Protein Expressing Cells H17188 Cheng Huang 1 C J Peters 1 2 and Shinji Makino 1 Departments of Microbiology and Immunology 1 and Pathology 2 The University of Texas Medical Branch at Galveston Galveston Texas 77555 1019 Received 20 October 2006 Accepted 23 February 2007 Analysis of severe acute respiratory syndrome coronavirus SCoV by either sucrose gradient equilibrium centrifugation or a virus capture assay using an anti SCoV S protein antibody demonstrated that the SCoV 6 protein which is one of the accessory proteins of SCoV was incorporated into virus particles Coexpression of the SCoV S M E and 6 proteins was sufficient for incorporation of the 6 protein into virus like particles Cells transfected with plasmid expressing the 6 protein released SCoV 6 protein however infected cells released SCoV 6 protein only in association with SCoV particles Severe acute respiratory syndrome coronavirus SCoV is known to be the causative agent of the global severe acute respiratory syndrome epidemic occurring during 2002 and 2003 1 8 9 The 3H11032 one third of the SCoV genome encodes the spike S membrane M and envelope E proteins all of which are envelope proteins the N protein which forms a helical nucleocapsid with the genomic RNA and the eight accessory proteins 3a 3b 6 7a 7b 8a 8b and 9b 10 15 19 Expression studies have uncovered some of the biological ac tivities of these accessory proteins 20 and yet their functions in infected cells and hosts are largely unexplored SCoV ac cessory proteins are dispensable for SCoV replication in cul tured cells 20 21 and the 3a 7a and 7b proteins are incor porated into virus particles 4 6 16 17 The 63 amino acid SCoV 6 protein is a membrane protein that is present mainly in the endoplasmic reticulum and Golgi compartments 3 14 proximal to the virus assembly site Past studies showed that SCoV lacking the gene encoding the 6 protein is viable 21 and a murine coronavirus expressing SCoV 6 protein has increased virulence in mice 14 The latter observation may be related to a finding that SCoV 6 protein expression accelerates murine coronavirus replication 18 Furthermore an expression study demonstrated that the SCoV 6 protein like the SCoV 3b and N proteins is an interferon antagonist 7 while further studies are needed to establish the biological functions of the 6 protein in SCoV infected cells To determine whether the SCoV 6 protein is a virion asso ciated protein we investigated the presence of the 6 protein in the purified SCoV particles Supernatants harvested from SCoV infected Caco2 cells were irradiated with 2 H11003 10 6 rads from a Gammacell 60 Co source model 109A J L Shepherd and Associates San Fernando CA and clarified by low speed centrifugation and filtration through a 0 45 H9262m syringe filter Then SCoV was purified by ultracentrifugation on a 20 to 60 continuous sucrose gradient at 26 000 rpm for 18 h using a Beckman SW28 rotor 4 5 Twelve fractions each consisting of 1 ml were collected and each was examined on Western blots using antibodies against the S M N and 6 proteins Fig 1 The rabbit anti SCoV 6 protein peptide antibody was pre pared by immunizing rabbits with the synthetic peptide N ELDDEEPMELDY corresponding to amino acids 51 to 62 of the 6 protein Commercially available anti S antibody IGM 541 IMGENEX and anti SCoV M antibody AP6008b Abgent were used Mouse anti N serum was provided by Xiao Hua Li at The University of Texas Southwestern Medical Center Dallas TX In agreement with our previous studies 4 5 the strongest signals for the S N and M proteins were detected in fractions 5 and 6 Fig 1 showing that the buoyant density of SCoV was between 1 18 and 1 20 g ml Most of the 6 protein signal was present in fraction 6 strongly suggesting the association of the 6 protein with virions The presence of the 6 protein in virions was further con firmed using a virus capture assay 4 5 Clarified virus samples were partially purified by centrifugation through 20 sucrose cushions at 26 000 rpm for3hinaBeckman SW28 rotor followed by immunoprecipitation with mouse monoclonal anti SCoV S protein antibody NR 617 NIH Biodefense and Emerging Infections Research Resources Repository or with an unrelated mouse monoclonal anti H2K antibody in the ab sence of detergent 11 12 We collected the pelleted SCoV particles the immunoprecipitated samples and the superna tant from the samples after initial anti SCoV S protein anti body mediated precipitation virus capture assay supernatant About 10 of the pelleted sample Fig 2A lane 1 ca 10 of the immunoprecipitated samples Fig 2A lanes 2 and 3 and 3 3 of the virus capture assay supernatant Fig 2A lane 4 were applied to the gel electrophoresis Western blot analysis demonstrated that both the 6 and the M proteins were present in the virions immunoprecipitated by anti S antibody Fig 2A lane 3 and showed that neither the 6 protein nor the M protein was detected in samples immunoprecipitated by the anti H2K antibody Fig 2A lane 2 Furthermore no 6 or M 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 H17188 Published ahead of print on 7 March 2007 5423 on March 16 2015 by UCSF Library IP sample obtained after immunoprecipitation 5424 NOTES J VIROL on March 16 2015 by UCSF Library we did not detect any amounts of the 6 protein that were not associated with SCoV particles Fig 2A lane 4 yet we detected the presence of 6 protein in the gradient purified SCoV particles Fig 2B and C The virus capture assay effi ciently excludes various cellular contaminants from the sam ples and allows preparation of highly purified virions from virally infected cells developing cytopathic effects this assay is used to demonstrate the specific incorporation of viral RNA and proteins into coronavirus particles 2 and host proteins into retrovirus particles 13 VLP incorporation of the 6 pro tein also supported the data that suggested that the SCoV 6 protein was virion associated These data highlight how un likely the possibility is that the bulk of the SCoV 6 protein in SCoV infected cell culture media was merely a part of the cellular debris rather it was part of the virion Because those experiments using SCoV were performed after irradiation of samples with 60 Co one might suspect that irradiation of the samples may have artificially caused an association of SCoV particles with released 6 protein which was not incorporated into SCoV particles However this possibility is highly unlikely because nearly all of the released 6 protein must have artifi cially bound to SCoV particles by irradiation to generate the data shown in Fig 1 and 2 In summary sucrose gradient purification of SCoV particles and the virus capture assay both convincingly pointed to the possibility that most of the 6 pro tein was released in association with SCoV particles from SCoV infected cells The present study and past studies revealed that SCoV ac cessory proteins 3a 6 7b and 7a are incorporated into SCoV FIG 3 Release of the SCoV 6 protein from cells expressing the 6 protein A 293T cells were transfected with empty vector pCAGGS mock or pCAGGS 6 6 At 48 h posttransfection supernatants from transfected cells were clarified applied onto a 20 sucrose cushion and centrifuged at 26 000 rpm for3hat4 C The pellets were sus pended in 1H11003 SDS PAGE loading buffer Medium Cell lysates were prepared with 1H11003 SDS PAGE loading buffer Cell Samples were separated on a 15 SDS PAGE gel and subjected to Western blot analysis with anti SCoV 6 protein antibody 6 The membranes were reprobed with anti actin antibody Actin B The supernatant of 293T cells expressing the 6 protein was collected 48 h after transfection and partially purified by centrifugation through a 20 sucrose cushion The pellets were suspended in NTE buffer loaded on a 20 to 60 continuous sucrose gradient and centrifuged at 26 000 rpm for 18 h using a Beckman SW28 rotor Twelve fractions were taken from the bottom and the densities for each action were measured upper panel Each fraction was then diluted at least threefold with NTE buffer and was then centrifuged through a 20 sucrose cushion The recovered pellets were suspended in 1H11003 SDS PAGE loading buffer and subjected to Western blot analysis with anti SCoV 6 protein an tibody lower panel FIG 4 Assembly of SCoV 6 protein into VLPs A mixture of plasmids containing 9 H9262g of pCAGGS S 6 H9262g of pCAGGS M 4 H9262gof pCAGGS E and 0 5 H9262g of pCAGGS 6 was transfected into 293T cells lanes 2 3 and 4 As a control cells were transfected with 19 5 H9262gof pCAGGS lane 1 At 48 h posttransfection culture media were har vested and the released VLPs were pelleted by centrifugation through a 20 sucrose cushion SCoV VLPs were resuspended in NTE buffer containing 0 3 of bovine serum albumin and immunoprecipitated with nonspecific monoclonal mouse anti H2K antibody lane 3 or mouse anti SCoV S protein monoclonal antibody NR 617 lane 4 The captured SCoV VLPs lanes 3 and 4 and cell lysates lanes 1 and 2 were analyzed using Western blot analysis with anti SCoV 6 protein antibody top panel and anti M protein antibody bottom panel VOL 81 2007 NOTES 5425 on March 16 2015 by UCSF Library CKM http jvi asm org Downloaded from particles 4 6 16 17 although these accessory proteins are secreted from infected cells and from expressing cells in dif ferent ways The 3a protein is released from both SCoV in fected cells and 3a expressing cells 5 The 7a protein is not released from infected cells or from 7a expressing cells 4 5 The 7b protein is not released from 7b expressing cells 16 while release of 7b protein from infected cells has not been examined yet The 6 protein was released from 6 protein expressing cells Fig 3 and yet sucrose gradient purification of SCoV Fig 1 and the virus capture assay Fig 2 both suggested that unlike the 3a protein the 6 protein alone was not released from SCoV infected cells We speculate that some viral envelope proteins may interact with the 6 protein thereby preventing its release and mediating its incorporation into SCoV particles in infected cells The report that an attenuated murine coronavirus express ing the SCoV 6 protein exhibits an enhanced virulence in mice 14 and promotes murine coronavirus replication in infected cells 18 points to the possible involvement of the 6 protein in SCoV pathogenesis and replication A recent expression study reported that the SCoV 6 protein is an interferon antagonist 7 How the 6 protein affects SCoV virulence is unclear yet the present 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