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10 1128 JVI 78 9 4552 4560 2004 2004 78 9 4552 DOI J Virol Jr and Donna M Ambrosino Gregory J Babcock Diana J Esshaki William D Thomas Receptor Protein Are Required for Interaction with Respiratory Syndrome Coronavirus Spike Amino Acids 270 to 510 of the Severe Acute http jvi asm org content 78 9 4552 Updated information and services can be found at These include REFERENCES http jvi asm org content 78 9 4552 ref list 1at This article cites 27 articles 14 of which can be accessed free CONTENT ALERTS more articles cite this article Receive RSS Feeds eTOCs free email alerts when new http journals asm org site misc reprints xhtmlInformation about commercial reprint orders http journals asm org site subscriptions To subscribe to to another ASM Journal go to on April 4 2014 by USC Norris Medical Library http jvi asm org Downloaded from on April 4 2014 by USC Norris Medical Library http jvi asm org Downloaded from JOURNAL OF VIROLOGY May 2004 p 4552 4560 Vol 78 No 9 0022 538X 04 08 00H110010 DOI 10 1128 JVI 78 9 4552 4560 2004 Copyright 2004 American Society for Microbiology All Rights Reserved Amino Acids 270 to 510 of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Are Required for Interaction with Receptor Gregory J Babcock Diana J Esshaki William D Thomas Jr and Donna M Ambrosino Massachusetts Biologic Laboratories University of Massachusetts Medical School Jamaica Plain Massachusetts 02130 Received 13 October 2003 Accepted 2 January 2004 A novel coronavirus severe acute respiratory syndrome coronavirus SARS CoV has recently been iden tified as the causative agent of severe acute respiratory syndrome SARS SARS CoV appears similar to other coronaviruses in both virion structure and genome organization It is known for other coronaviruses that the spike S glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane Here we describe the construction and expression of a soluble codon optimized SARS CoV S glycoprotein comprising the first 1 190 amino acids of the native S glycoprotein S 1190 The codon optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system S 1190 binds to the surface of Vero E6 cells a cell permissive to infection as demonstrated by fluorescence activated cell sorter analysis suggesting that S 1190 maintains the biologic activity present in native S glycop rotein This interaction is blocked with serum obtained from recovering SARS patients indicating that the binding is specific In an effort to map the ligand binding domain of the SARS CoV S glycoprotein carboxy and amino terminal truncations of the S 1190 glycoprotein were constructed Amino acids 270 to 510 were the minimal receptor binding region of the SARS CoV S glycoprotein as determined by flow cytometry We speculate that amino acids 1 to 510 of the SARS CoV S glycoprotein represent a unique domain containing the receptor binding site amino acids 270 to 510 analogous to the S1 subunit of other coronavirus S glycoproteins Severe acute respiratory syndrome SARS is a recently de scribed disease that has affected approximately 8 500 people worldwide with a mortality rate of approximately 10 accord ing to the World Health Organization The causative agent of SARS is a newly identified coronavirus SARS CoV first iso lated by propagation on Vero E6 cells 5 12 17 The SARS CoV genome has been sequenced and the probable coding regions for viral proteins have been deduced Like other coro naviruses SARS CoV is a positive strand RNA virus that en codes four main structural proteins M N E and S 20 Genetic analysis of the coding regions has demonstrated that SARS CoV is distinct from the three known antigenic groups of coronaviruses 5 12 however recent data studying the replicase gene suggest that SARS CoV may be most related to group 2 coronaviruses 21 The S glycoprotein a 1 255 amino acid type I membrane glycoprotein 20 is the prominent protein present in the viral membrane and presents as the typical spike structure found on all coronaviruses SARS CoV S glycoprotein domain structure has been deduced from sequence analysis 20 The S glyco protein consists of a leader amino acids 1 to 14 an ectodo main represented by amino acids 15 to 1190 a membrane spanning domain amino acids 1191 to 1227 and a short intracellular tail amino acids 1227 to 1255 20 The full length SARS CoV S glycoprotein has 23 potential N linked glycosylation sites predicted by sequence analysis 20 For group 2 and group 3 coronaviruses the S glycoprotein is post translationally cleaved into two noncovalently associated sub units S1 and S2 6 15 22 23 The motif that leads to cleavage of the subunits in these coronaviruses 15 is not present in SARS CoV suggesting that cleavage of the SARS CoV S gly coprotein does not occur 20 Although the process by which SARS CoV penetrates the cellular membrane has not been determined the mechanism is most likely similar to that described for other coronaviruses The S glycoprotein interacts with the cellular surface and for coronaviruses HCoV 229E and mouse hepatitis virus MHV amino acids 1 to 547 2 and 1 to 330 13 respectively are required for binding to the cellular receptor This interaction is predicted to lead to conformational changes in the carboxy terminal half of the S glycoprotein This change culminates in fusion of the virus and host cell membranes allowing for entry of the virus 25 27 Sequence analysis of the SARS CoV S glycoprotein using the LearnCoil VMF software has predicted the presence of two coiled coil motifs present at amino acids 900 to 974 and 1148 to 1190 These coiled coil structures are present in the fusion domain of many varied viruses including MHV 4 11 14 and human immunodeficiency virus type 1 9 of which entry events have been predicted to occur as described above Here we describe the construction and expression of a codon optimized gene encoding the soluble ectodomain amino acids 1 to 1190 of the SARS CoV S glycoprotein Corresponding author Mailing address Massachusetts Biologic Laboratories University of Massachusetts Medical School 305 South St Jamaica Plain MA 02130 Phone 617 983 6415 Fax 617 983 6477 E mail greg babcock umassmed edu 4552 on April 4 2014 by USC Norris Medical Library http jvi asm org Downloaded from Codon optimized S glycoprotein S 1190 was secreted into the growth medium and purified by affinity chromatography Ex pression levels of secreted S 1190 glycoprotein were determined to be approximately 5 mg liter after purification The S 1190 synthetic S glycoprotein was shown to have an apparent mo lecular mass of 170 kDa a size similar to that observed for native S protein expressed in SARS CoV infected Vero E6 cells Purified S 1190 protein was readily detected by human SARS convalescent phase serum provided by Larry Ander son Centers for Disease Control and Prevention CDC as determined by Western blot analysis Synthetic S glycoprotein could also bind to the surface of Vero E6 cells demonstrating that soluble codon optimized S glycoprotein retains the bio logic activity present in the native molecule Carboxy terminal truncations of S 1190 were produced and it was demonstrated that the amino acids 1 to 510 S 510 are required for binding to Vero E6 cell surfaces Amino terminal truncations of the S 510 glycoprotein demonstrated that amino acids 270 to 510 contain the minimal receptor binding domain of the SARS CoV S glycoprotein MATERIALS AND METHODS Construction of a synthetic gene encoding soluble codon optimized SARS CoV spike S protein and S protein fragments The amino acid sequence of the SARS CoV Urbani strain S protein was obtained from the NCBI database AAP13441 The soluble portion of the protein was determined to be the first 1 190 amino acids of 1 255 and as such only the DNA encoding this sequence was synthesized The DNA sequence was codon optimized for mammalian cell expression 1 16 replacing the natural codons with the following optimum codons alanine GCC arginine CGC asparagine AAC aspartic acid GAC cysteine TGC glutamic acid GAG glutamine CAG glycine GGC histidine CAC isoleucine ATC leucine CTG lysine AAG me thionine ATG phenylalanine TTC proline CCC serine TCC threonine ACC tryptophan TGG tyrosine TAC and valine GTG Runs of Cs and Gs were avoided to simplify both synthesis of oligonucleotides as well as PCR conditions When these stretches of Gs and Cs occurred suboptimal codons were used The 5H11032 end of the gene was modified to include a restriction site for HindIII and an irrelevant upstream overhang to facilitate cloning The 3H11032 end of the synthetic gene was similarly modified to include an XbaI site and overhang sequences A total of 104 oligonucleotides were obtained Integrated DNA Technologies polyacrylamide gel electrophoresis purified that represented the entire coding region of both the sense and antisense strands of the S protein gene as well as engineered restriction sites The most 5H11032 oligonucleotide of each strand was a 35 mer and all others were 70 mers resulting in a 35 bp overlap between strands In essence the oligonucleotides from the sense strand fully overlapped the oligonucleotides of the antisense strand leaving no gaps Construction of the codon optimized gene was performed as follows Thirteen groups of oligonucle otides were selected that contained eight oligonucleotides four sense and four antisense in each group PCR was performed on each set in a reaction mixture containing 20 H9262M deoxynucleoside triphosphates 30 pmol of end oligonucleo tides 10 pmol of internal oligonucleotides 1H11003 cloned Pfu reaction buffer Strat agene and1UofTurbo Pfu Stratagene Thirty cycles of thermocycling 95 C for 15 s 62 C for 30 s and 68 C for 2 min were performed and the PCR products were resolved on 1 agarose gels Specific products were gel purified Qiagen and divided into four separate groups containing either three or four of the first step PCR products PCR was again performed on each group using oligonucleotides corresponding to the most 5H11032 end of each strand These four PCR products were resolved on 0 8 agarose gels and gel purified as before The four PCR products were mixed and amplified using oligonucleotides correspond ing to the 5H11032 end of each strand of the entire synthetic gene This final amplifi cation yielded the 3 605 bp sequence consisting of the synthetic gene flanked by restriction sites The final PCR product encoding the SARS CoV S glycoprotein gene was digested with HindIII and XbaI and cloned into pcDNA3 1 Myc His Invitrogen in frame with the c myc and His 6 epitope tags The cloned gene was sequenced to confirm that no errors had been accumulated during the PCR process Of the four clones sequenced none had sequence errors and no further genetic manip ulations were required Once the sequence of the full length soluble SARS CoV S glycoprotein gene was confirmed DNA encoding carboxy terminally truncated soluble S glycopro teins was synthesized by PCR amplifying the desired fragment from the vector containing the full length codon optimized gene encoding the S glycoprotein Since the codon optimized S 1190 gene was used as a template for PCR all truncated constructs were also codon optimized Truncations were then cloned into pcDNA3 1 Myc His as described above and the DNA sequence was con firmed FIG 1 Expression and purification of soluble S 1190 glycoprotein Codon optimized S 1190 glycoprotein was cloned into pcDNA3 1 Myc His and expressed in HEK 293T 17 cells to a level of 5 mg liter Protein was purified using metal affinity chromatography dialyzed and concentrated Purified protein was analyzed by Coomassie staining A or Western blotting using the anti c myc antibody for detection B FIG 2 The apparent molecular mass of the S 1190 protein is similar to that of native S protein S 1190 protein 200 ng Vero E6 cell extract and SARS CoV infected Vero E6 cell extract were resolved by SDS PAGE Proteins were transferred to a solid support and Western blotting was performed using either human SARS convalescent phase serum top panel or mouse anti synthetic S protein bottom panel The major species detected for S 1190 and SARS infected extract in both cases was of an apparent molecular mass of approximately 170 kDa No signal was observed in the lane containing the Vero E6 cell extract VOL 78 2004 SARS CoV SPIKE RECEPTOR BINDING DOMAIN 4553 on April 4 2014 by USC Norris Medical Library http jvi asm org Downloaded from N terminal truncations were also synthesized PCR was used to amplify the leader sequence of the S 1190 gene containing a 3H11032 overhang corresponding to downstream sequences The downstream sequences were then amplified and combined with the leader overhang PCR product PCR was again performed to synthesize copies of a gene that consisted of the S 1190 leader fused immediately 5H11032 of the downstream coding region These constructs essentially created dele tions between the leader peptide and the desired downstream sequence Cells and cell culture HEK 293T 17 and Vero E6 cells obtained from the American Type Culture Collection were grown in Dulbecco s modified Eagle s medium DMEM supplemented with 10 fetal bovine serum and 100 IU of penicillin streptomycin complete DMEM at 37 C with 5 CO 2 To harvest cells phosphate buffered saline PBS containing 5 mM EDTA was added to the tissue culture dish and incubated for 5 min at room temperature Expression and purification of codon optimized S glycoproteins All con structs were transfected into HEK 293T 17 cells using Lipofectamine 2000 In vitrogen as described by the manufacturer Briefly cells were grown to 80 confluence in 150 mm tissue culture dishes in 15 ml of DMEM 10 fetal calf serum FCS Thirty micrograms of DNA mixed with 75 H9262l of Lipofectamine 2000 was added to the cells and plates were incubated overnight at 37 C Medium was removed and stored and fresh complete DMEM was added to the cells Cells were incubated for an additional 24 h at which time 3 mM sodium butyrate Sigma was added to the medium An additional 24 h incubation was performed and supernatants were removed from the plate This supernatant was combined with the transfection supernatant and filtered using a 0 45 mm pore size filter apparatus Filtered supernatants were mixed with Ni nitrilotriacetic acid agarose Invitrogen at a ratio of 0 5 ml of agarose for 40 ml of culture supernatant Supernatant agarose mixtures were incubated for2honarocking platform at room temperature Agarose was removed from the supernatant by column filtration Beads were washed with PBS and protein was eluted using 250 mM imidazole Eluted protein was dialyzed against PBS for2hatroom tem perature and concentrated to 2 ml with an Amicon Centriprep YM 10 Sodium dodecyl sulfate PAGE SDS PAGE and Coomassie blue staining were used to determine purity of isolated proteins SDS PAGE and Western blotting Various concentrations of purified S glyco proteins were mixed with 2H11003 reducing Laemmli sample buffer and boiled for 5 min Samples were resolved using 12 Novex gels Invitrogen for 1 5 h at 200 V Gels were transferred to Immobilon P Millipore as described by the man ufacturer and Western blot analysis was performed Proteins were detected using the anti c myc 9E10 antibody 0 1 H9262g ml Sigma followed by an anti mouse immunoglobulin G IgG horseradish peroxidase conjugate 1 5 000 Jackson ImmunoResearch For detection with human convalescent phase se rum provided by Larry Anderson CDC a dilution of 1 2 000 was used followed by detection with anti human IgG horseradish peroxidase Jackson ImmunoRe search For detection with mouse serum raised against synthetic S glycoproteins the method was as described for the anti c myc antibody Membranes were incubated with enhanced chemiluminescence reagent for 1 min and exposed to X Omat AR film for various periods of time S glycoprotein binding assay Vero E6 or HEK 293T 17 cells were harvested with PBS 5 mM EDTA and aliquoted to microcentrifuge tubes 1 H11003 10 6 to 5 H11003 10 6 each Pellets were resuspended in PBS containing 10 fetal bovine serum and various concentrations of the truncated soluble S glycoproteins 0 01 nM to 1 H9262M Cells and S glycoprotein were incubated for1hatroom temperature and washed once in PBS 2 FCS Pellets were resuspended in 100 H9262lofPBS 2 FCS containing 10 H9262g of anti c myc 9E10 antibody ml incubated for1hat4 H9265 C and washed once in PBS 2 FCS Pellets were resuspended in 100 H9262lof PBS 2 FCS containing 5 H9262l of anti mouse IgG phycoerythrin PE Jackson ImmunoResearch Mixtures were incubated at 4 C for 40 min and washed twice and fluorescence activated cell sorter FACS analysis was performed using a FACScan instrument with CellQuest software Becton Dickinson In order to specifically block S glycoprotein binding to Vero E6 cells human convalescent phase serum was incubated with cells and S glycoprotein Serum concentration never exceeded 10 and as human serum was diluted FCS was used to normalize all reaction mixtures to a final concentration of 10 serum Normal human serum was used as a negative control RESULTS Construction and expression of soluble codon optimized SARS CoV S glycoprotein The genes that encode viral pro teins quite often have poor codon usage leading to difficulties in producing sufficient quantities of purified recombinant pro tein 8 To overcome the possible issue of poor codon usage of the S glycoprotein gene we constructed a synthetic codon optimized S glycoprotein gene Analysis of optimal codon us age in mammalian cells has been described elsewhere 1 16 A codon optimized gene encoding the first 1 190 amino acids of the SARS CoV S glycoprotein S 1190 was synthesized and cloned into the mammalian expression vector pcDNA 3 1 Myc His The first 1 190 amino acids represent the predicted leader sequence and extracellular domain of the S glycoprotein ex cluding transmembrane and intracellular domains As such when expressed the gene product is a secreted soluble version of the S glycoprotein The vector used contains two epitope tags the c myc and His 6 tags The c myc tag was exploited for FIG 3 S 1190 specifically binds to the surface of Vero E6 cells A Vero E6 cells diamonds or HEK 293T 17 cells squares were incubated with 1 to 100 nM S 1190 protein to determine the ability to bind to cell surfaces S 1190 binding was detected by anti c myc antibody followed by anti mouse IgG PE Samples were analyzed by flow cy tometry and mean fluorescence intensity was plotted B Vero E6 cells were incubated with 30 nM S 1190 glycoprotein in the presence of increasing concentrations of normal circles or SARS convalescent phase squares human serum Serum concentration was maintained at 10 by the addition of fetal bovine serum Binding was measured by flow cytometry and the results were plotted as a percentage of the signal observed in a sample containing no test serum 4554 BABCOCK ET AL J VIROL on April 4 2014 by USC Norris Medical Library http jvi asm org Downloaded from immunoprecipitations and Western blot analysis of proteins while the His 6 tag allowed for native purification of expressed protein pcDNA 3 1 Myc His S 1190 was transfected into HEK 293T 17 cells supernatants were recovered and S 1190 glyco protein was purified by metal affinity chromatography Pro teins were eluted from the resin with imidazole dialyzed and concentrated S 1190 concentration was determined by both spectrophotometry and bicinchoninic acid both of which yielded equivalent results data not shown It was determined that secreted S 1190 was expressed at a level of approximately 5 mg liter after purification To assess purity of the S 1190 glycoprotein preparations pro teins were resolved by SDS PAGE and visualized by Coomas sie staining Fig 1A The major band of a relative molecular mass of 170 kDa was observed