【病毒外文文献】2005 The severe acute respiratory syndrome coronavirus 3a is a novel structural protein

Chao Hong ia Sinica Products February binding to host receptors and membrane fusion 3 known as X1 in 10 asORF3in 11 and as U274 in 12 13 is the largest of these unique ORFs and consists of 274 amino acids Three groups have independently re ported the expression of 3a in SARS CoV infected cells 13 15 and it was also detected in a SARS CoV infected Corresponding author Fax 65 67791117 E mail address mcbtanyj imcb a star edu sg Y J Tan 1 These authors contributed equally to this work Biochemical and Biophysical Research Communica 0006 291X see front matter C211 2005 Elsevier Inc All rights reserved The recent severe acute respiratory syndrome SARS epidemic which a ected over 30 countries resulted in more than 8000 cases of infection and more than 800 fatalities World Health Organization http www who int csr sars country en A novel coronavi rus was identified as the etiological agent of SARS 1 Like all coronaviruses the SARS CoV genome encodes for four major structural proteins nucleocapsid N spike S membrane M and envelope E The RNA is packaged by the N protein into a helical nucleocapsid 2 The S protein which forms morphologically charac teristic projections on the virion surface mediates The M protein is a triple spanning integral membrane protein with a short ectodomain and a large carboxyl terminus endodomain 4 More recently the E protein was showed to play a major role in coronavirus assem bly 5 8 Another structural protein the hemagglutinin esterase glycoprotein HE is found in only a subset of coronaviruses but its role in the virus life cycle has not been well established 9 The SARS CoV genome does not appear to encode for a HE protein 10 In addition the SARS CoV genome also contains an other nine open reading frames ORFs with no homo logue in other known coronaviruses 10 11 3a also Abstract The severe acute respiratory syndrome coronavirus SARS CoV 3a protein is one of the opening reading frames in the viral genome with no homologue in other known coronaviruses Expression of the 3a protein has been demonstrated during both in vitro and in vivo infection Here we present biochemical data to show that 3a is a novel coronavirus structural protein 3a was detected in virions purified from SARS CoV infected Vero E6 cells although two truncated products were present predominantly instead of the full length protein In Vero E6 cells transiently transfected with a cDNA construct for expressing 3a a similar cleavage was observed Furthermore co expression of 3a membrane and envelope proteins using the baculovirus system showed that both full length and truncated 3a can be assembled into virus like particles This is the first report that demonstrated the incorporation of 3a into virion and showed that the SARS CoV encodes a novel coronavirus structural protein C211 2005 Elsevier Inc All rights reserved Keywords Severe acute respiratory syndrome SARS coronavirus 3a viral protein Structural protein Baculovirus Virus like particles The severe acute respiratory is a novel structural Shuo Shen a 1 Pi Shiu Lin b 1 Yu Chan Seng Gee Lim a Wanjin a Institute of Molecular and Cell Biology 61 Biopolis b Institute of Molecular Biology Academ c Wuhan Institute of Biological Received 18 doi 10 1016 j bbrc 2005 02 153 syndrome coronavirus 3a protein b Aihua Zhang c Xiaoming Yang c a Yee Joo Tan a Drive Proteos Singapore 138673 Singapore Nankang Taipei 115 Taiwan ROC Wuhan 430060 PR China 2005 tions 330 2005 286 292 BBRC Here we provide biochemical evidence showing that S Shen et al Biochemical and Biophysical Research Communications 330 2005 286 292 287 3a is a novel coronavirus structural protein First 3a is not only secreted into the culture supernatant of SARS CoV infected cells but it can also be detected in purified virion Second co expression of 3a with M and E in the baculovirus system resulted in the incorpo ration of 3a into viral like particles Similar to other cor onaviruses 5 7 the co expression of SARS CoV M and E in the baculovirus system was su cient for the formation of viral like particles 22 23 Our data showed that 3a is a novel coronavirus structural protein that is not essential for virus assembly but is incorpo rated into the virion when present Materials and methods Construction of plasmids The cDNA construct for expressing SARS CoV 3a protein was obtained from SARS CoV 2003VA2774 an isolate from a SARS patient in Singapore as previously described 13 pXJ3 0 3a for transient expression in mammalian cells was previ ously described 13 while pBacPAK8 3a used for the production of recombinant baculovirus was constructed for this study using the method previously described 22 Transient transfection of Vero E6 culture and infection with SARS CoV Vero E6 African green monkey kidney epithelial cells were purchased from the American Type Culture Collection and cultured as previously described 13 SARS CoV isolate from a SARS patient in China was used to infect Vero E6 cells as previously described 13 21 For metabolic labeling monolayer of cells was infected at multiplicity of infection of 0 1 for 2 h then starved with methionine Met and cysteine Cys free medium DMEM NEN for 0 5 h and were then labeled for 12 h by replacing with the medium supplemented with 35 S Met and 35 S Cys 100 Ci ml NEN At approximately 14 h post infection the cells showed approximately 50 cytopathic e ects CPE and the culture supernatant was collected Lysis bu er was added to the culture supernatant to give a final concentration of 50 mM Tris HCl pH 8 150 mM NaCl 0 5 NP40 BDH Laboratory Supplies 0 5 sodium deoxycholate and 0 005 SDS 1 lysis bu er The patientC213s lung specimen 14 Antibodies against 3a were also found in convalescent patients 12 14 16 3a was localized in the perinuclear region and was also trans ported to the cell surface where it could undergo inter nalization 13 14 It is tempting to postulate that 3a is a structural protein as only the coronavirus structural proteins S HE and E have been shown to be trans ported to the plasma membrane cell surface 17 21 3a can also interact with M and E which are two key players in the viral assembly of coronaviruses as well as with the S protein 13 15 hence it may be important for viral assembly and or release of virus from infected cells Indeed Zeng et al 15 detected disulfide linked com plexes of S and 3a in the culture supernatant of SARS CoV infected cells indicating that 3a could be se creted together with S possibly through the formation of virions However it is necessary to confirm this find ing with highly purified SARS CoV virions as viral pro teins could also be released into the culture supernatant through cell lysis remaining cells were lysed in 1 lysis bu er Then both cell lysates and culture supernatant were subjected to immunoprecipitation with rabbit polyclonal anti 3a antibody and protein A Sepharose beads Roche Molecular Biochemicals as previously described 13 The immuno complexes were separated on 15 SDS PAGE and detected by autoradiography as previously described 24 25 A mock infection was performed in parallel as negative control For detection of 3a by Western blot analysis a similar protocol was used except that no metabolic labeling was performed The cell lysates were subjected to Western blot as previously described 13 For transient expression of 3a Vero E6 cells were transfected with pXJ3 0 3a using Lipofectamine reagent Invitrogen according to the manu facturerC213s protocol Primary antibodies for Western blot analysis anti 3a rabbit polyclonal antibody 13 or anti tubulin monoclonal antibody Sigma were used at 1 3000 dilutions Quantification of autoradiographs was performed using a model Bio Rad GS 700 Imaging Densitometer with Bio Rad Multi Analyst version 1 02 Mac software Purification of virions from culture supernatant of SARS CoV in fected Vero E6 cells In order to obtainpurifiedvirion fromSARS CoV infected cells Vero E6 were infected as described above except that in order to obtain the highest virus titer the cells were left for C2448 h post infection when the CPE observed was C2490 Then b propiolactone was added to infected cell culture to a final concentration of 0 05 to inactivate infectivity The inactivation was examined by titration of treated samples in Vero E6 cells The viruses were harvested by freez ing thawing three times and cell debris was removed by centrifugation at 5000 rpm for 10 min Ultrafiltration was performed to concentrate viruses 300 000 NMWL Millipore The concentrated sample was applied to Sepharose 4B fast flow column Pharmacia following manufacturerC213s instruction The eluted fractions were examined by transmission electron microscopy and the fraction containing virus particles was used for Western blot analysis Insect cell culture and production of recombinant baculoviruses The Spodoptera frugiperda IPLB Sf21 Sf21 insect cell line was cultured as a monolayer in TNM FH insect medium containing 8 heat inacti vated fetal bovine serum as described previously 26 It was used for the propagation and infection of recombinant baculoviruses titers of viruses were determined by a newly developed quantitative real time PCR based method 27 Recombinant baculoviruses carrying the M and E genes were produced in a previous study 22 Here recombinant baculoviruses carrying the 3a gene were generated using Autographa californica multiple nucleopolyhedrovirus AcMNPV viral genome in a similar manner Briefly pBacPAK8 3a was co transfected with linearized viral DNA BaculoGold BD Biosciences by using Lipofectin Invitrogen into insect cells and successful recombinants were isolated by the indication of red fluorescence The recombinant baculoviruses encoding the E M and 3a proteins assigned as vABhRpE vABhRpM and vABhRp3a respectively were obtained by two or three round serial dilutions and all viral stocks were prepared and manipulated according to the standard protocol described by OC213Reilly et al 28 Isolation of coronavirus like particles VLPs Sf21 insect cells were co infected with vABhRpE vABhRpM and vABhRp3a at a multi plicity of infection of 1 5 1 At 4 days post infection cells were col lected by a cell scraper Costar and then re suspended in Tris bu ered saline TBS containing a cocktail of protease inhibitors 1 1000 dilution SET III Calbiochem and lysed by sonication The post nuclear supernatant was obtained by centrifugation at 1000 rpm for 10 min and was then placed on a 30 w w sucrose cushion for centrifugation at 34 000 rpm for 20 h Pellets were washed twice with TBSs resuspended in the same bu er and then subjected to a 20 60 w w sucrose gradient at 34 000 rpm for 60 h Twenty eight fractions monitored at a wavelength of 280 nm were extracted from the cen trifuged sucrose gradient using a density gradient fractionation system ISCO Each fraction of sucrose gradient was separated on 12 SDS PAGE and then transferred onto a PVDF membrane Immobilin P Millipore The membrane was blocked with 5 non fat milk for 1 h and then probed at a dilution of 1 5000 by antiserum at 4 C176C over night The antibody used to detect the 3a protein has been described previously 13 29 and was developed by immunizing rabbits with bacterially expressed recombinant proteins And the antibody for detecting the M protein was purchased from Abgent The antibody used to detect the E protein has been described previously 22 and was raised by immunizing rabbits with a synthetic peptide After three washes with 0 1 Tween 20 containing phosphate bu ered saline TPBS a horseradish peroxidase HRP conjugated goat anti rabbit IgG was added at a dilution of 1 2500 for 1 h at room temperature The blot was then washed with TPBS four times followed by visual ization using chemiluminescent reagent Western Lightning Perkin Elmer and developed on an X ray film BioMax Kodak Results and discussion SARS CoV 3a was secreted into the culture supernatant of infected Vero E6 cells and was incorporated into virions In order to determine if the SARS CoV 3a protein can be secreted cell lysates and culture supernatants from metabolically labeled SARS CoV infected Vero E6 were subjected to immunoprecipitation with a poly clonal anti 3a antibody As shown in Fig 1A a spe cific protein band of C2435 kDa was detected in cell lysates obtained from SARS CoV infected cells but not in cell lysates from mock infected cells lanes 1 and 2 Therefore 3a was expressed in the SARS CoV infected cells which is consistent with previous studies 30 The 3a protein was also specifically immu noprecipitated from the culture supernatant of SARS CoV infected cells Fig 1A lanes 3 and 4 indicating that 3a protein was secreted into the culture superna tant Consistently Zeng et al 15 recently reported the detection of disulfide linked complexes of S and 3a in the culture supernatant of SARS CoV infected cells Four other proteins with molecular weights less than 35 kDa in the cell lysates also co immunoprecip itated with 3a Fig 1A lane 2 The sizes of these pro teins are C2424 C2420 C2415 and C248 kDa and they could be host or viral proteins that bound to 3a or they could be cleaved degraded forms of 3a The smallest of these co immunoprecipitated proteins C248 kDa and another protein of C2410 kDa that co immunopre cipitated with 3a from the culture supernatant have similar molecular weight to that predicted for the E protein Fig 1A lanes 2 and 4 As the interaction be tween 3a and E has been previously demonstrated 13 virions infected with using 288 S Shen et al Biochemical and Biophysical Research Communications 330 2005 286 292 Fig 1 Expression of 3a in SARS CoV infected Vero E6 cells purified culture supernatants from mocked infected lanes 1 and 3 or SARS CoV anti 3a polyclonal antibody The 3a protein s or proteins that interacted lanes 2 and 4 but not in mocked infect cells B Western blot analysis in cells lysates obtained from mock infected cells lane 1 SARS CoV infected lane 3 Cells transiently transfected with a DNA construct for expressing to Western blot analysis lanes 4 and 5 Untransfected Vero E6 was include for each samples were verified by measuring the level of endogenous tubulin lane 3 and transfected cells A 35 S metabolically labeled cell lysates or cells lanes 2 and 4 were subjected to immunoprecipitation with 3a were detected in SARS CoV infected cells marked by arrowheads specific anti 3a antibody top panel to detect for the expression of 3a cells lane 2 and in virions purified from SARS CoV infected cells 3a were harvested at 1 or 2 days post transfection and subjected d as negative control lane 6 The amounts of total cell lysates loaded bottom panel Note that no tubulin was present in the purified virion phase of infection and that 3a is sensitive to cleavage over time Unfortunately it was not possible to repeat the purification of virion from an earlier phase of infec tion as the incidents of laboratory acquired SARS CoV infections 32 have led to a restriction on unnecessary protocols like purification of virions that requires high virus titer and specialized instrumentations using live SARS CoV SARS CoV 3a was incorporated into virus like particles in insect cells co infected with recombinant baculoviruses expressing the E M and 3a To express SARS CoV 3a protein in the baculovirus insect system recombinant virus vABhRp3a was gener ated as previously described 22 Expression of 3a in Sf21 insect cells after infection with vABhRp3a was ver ified by Western blot analysis Fig 2 Interestingly both the full length 3a and a truncated 3a product of C2424 kDa were specifically observed in the infected insect cells lane 1 but not in the control cells lanes 2 and 3 The cells were harvested at 4 days post infection and l Research Communications 330 2005 286 292 289 it is likely that the E protein C248 and C2410 kDa in the cell lysate and culture supernatant respectively ex pressed in the infected cells has co immunoprecipitated with 3a Interestingly the relative amount of E to 3a in the immuno complexes from culture supernatant was significantly higher when compared to the cell lysate suggesting that 3a may need to be complexed with E before secretion out of the cells whilst there was a pool of free 3a intracellularly The molar ratios of E 3a as computed by quantification of the autoradio graph shown in Fig 1A were 3 2 in the culture super natant and 7 8 in the cell lysate Based on this assumption we estimated that the molar ratio of E to 3a in the virion is 3 2 It should be noted that this is only an approximation and future analysis of highly purified virus samples by immunoprecipitation with specific monoclonal antibodies as recently described for transmissible gastroenteritis coronavirus by Escors et al 31 will be required to determine an accurate stoichiometric ratio of 3a in the virion relative to E and the other structural proteins Next the presence of 3a in purified virion was deter mined in order to confirm if 3a was secreted as part of a virion Fig 1B Due to the highly contagious nature of the SARS CoV it was not feasible to subject SARS CoV to the standard sucrose gradient purifica tion Instead the virus was subjected to size exclusion chromatography and the presence of virion in the eluted fractions was confirmed by electron microscopy studies data not shown As this was performed as part of a vaccine development program at the Wuhan Institute of Biological Products China infected cells were col lected at late infection 90 CPE in order to obtain the highest titer of virus Western blot analysis showed that two truncated forms of 3a C2424 and C2420 kDa were detected in purified virion using specific anti 3a antibody Fig 1B lane 3 These two truncated forms of 3a may correspond to the two protein bands of the similar sizes observed in Fig 1A lane 2 Curiously the full length 3a was not easily detectable although it was clear that the full length 3a was expressed in and secreted from the infected cells at earlier phase of infection 50 CPE Fig 1B lane 2 and results above No cellular tubulin which is highly abundant in the cell lysates was associated with the purified virion Fig 1B lanes 1 3 indicating that the purification process successfully eliminated proteins that were not associated with the virion In order to understand the time course for the cleav age of 3a Vero E6 cells were transiently transfected with a cDNA construct to express 3a Fig 1B lanes 4 and 5 The results showed that 3a can indeed be cleaved into smaller products by 2 days post transfection Col lectively the probable explanation for our observation of only truncated forms of 3a in the virion is that the S Shen et al Biochemical and Biophysica virion was purified from infected cells at a much later Fig 2 Expression of 3a in insect cells Sf21 insect cells were infected with recombinant baculovirus carrying both 3a and DsRed2 reporter gene genes lane 1 Cells were harvested at 4 days post infection lysed and the cell lysate was subjected to Western blot analysis using anti 3a antibody For negative controls the cell lysates from Sf21 cells infected with recombinant baculovirus carrying only DsRed2 gene lane 2 or mock infected Sf21 cells lane 3 were used therefore the amount of the truncated 3a was high sim ilar to what was observed in Vero E6 cells 2 days post transfection with pXJ 3a construct Fig 1B Hence similar processing of 3a was observed in both mamma lian and insect cells Previously the formation of VLPs in insect cells expressing the SARS CoV structural proteins M and E was demonstrated 22 23 The S protein could also be incorporated into VLPs when it is expressed together with M and E 22 23 In order to determine if SARS CoV 3a protein can be incorporated into VLPs insect cells co expressing 3a M and E were subjected to su crose gradient centrifugation followed by Western blot analysis As shown in Fig 3A the 3a protein was found mainlyinthefractions17and18 Thesamefractionsalso contained the majority of the M and E proteins Figs 3B and C Previously electron micros