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E cient assembly and release of SARS a heterologous expression Eduardo Mortola a a Department of Infectious and Tropical Diseases London School of Hygiene University 18 September The expression level size and authenticity of each recombinant SARS CoV protein were determined In addition immunofluo ratory disease for which an e ective and safe vaccine is required 2 SARS CoV is phylogenetically distinct from other leads to the formation of the helical nucleocapsid The M protein is associated with specialized intracellular membrane structures and interactions between the M and E proteins and coronavirus membrane associated proteins such as M E and SE mail address polly roy lshtm ac uk P Roy FEBS Letters 576 2004 174 178 FEBS 28839 californica Sf9 Spodoptera frugiperda WB Western blotting EM electron microscopy PI post infection proteins of SARS at a high level by using the heterologous insect cell protein expression system that was used to generate BTV VLPs could be a challenging task in comparison to non envelope BTV VLPs Abbreviations SARS CoV severe acute respiratory syndrome associ ated coronavirus VLPs virus like particle AcNPV Autographa characterized coronaviruses 3 The genome is composed of a relatively conserved region encoding an RNA dependent RNA polymerase and a variable region containing open reading frames encoding sequences for viral structural proteins spike S envelope E membrane M and nucleocapsid N 4 5 Like some other coronaviruses the variable region of SARS nucleocapsids result in budding through the membrane 6 The S protein is incorporated into the viral envelope again by interaction with M and mature virions are released from the plasma membrane 15 Assembly of coronavirus capsid proteins by a heterologous mammalian expression system has been reported for mouse hepatitis virus 16 18 and the transmissible gastroenteritis virus of swine 19 However assembly and expression of three Corresponding author Fax 44 0 207 7927 2839 rescence and FACS analysis confirmed the cell surface expres sion of the S protein Co infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs On the other hand simultaneous high level expression of S E and M by a single recombinant virus allowed the very e cient assembly and release of VLPs These data demonstrate that the VLPs are morpho logical mimics of virion particles The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus o ers a potential candidate vaccine for SARS C211 2004 Federation ofEuropean Biochemical Societies Published by Elsevier B V All rights reserved Keywords Virus like particle SARS Baculovirus system 1 Introduction The severe acute respiratory syndrome SARS associated coronavirus SARS CoV also named infectious atypical pneumonia is a newly emergent member in the family Coro naviridae 1 SARS CoV causes a transmissible febrile respi b Division of Geographic Medicine Department of Medicine Received 20 July 2004 revised 27 August Available online Edited by Hans Diete Abstract Virus like particles VLPs produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome SARS control In this study using the baculovirus system we generated recombinant viruses that expressed S E M and N structural proteins of SARS CoV either individually or simultaneously 0014 5793 22 00 C211 2004 Federation of European Biochemical Societies Published doi 10 1016 j febslet 2004 09 009 coronavirus like particles by system Polly Roy a b and Tropical Medicine Keppel Street London WC1E 7HT UK of Alabama at Birmingham AL 35294 USA 2004 accepted 8 September 2004 2004 r Klenk CoV contains additional open reading frames that encode putative non structural proteins whose function is largely un known 6 In the Coronaviridae family the envelope M protein is a triple spanning membrane glycoprotein that interacts with the nucleocapsid and S protein during assembly 7 Another component essential in the assembly process is the small E protein This protein is generally a minor virion constituent It is largely embedded within the viral membrane and only its hydrophilic carboxy terminus protrudes inside the virion 8 9 The spike S protein which is the second most abundant envelope protein is a large type I transmembrane glycoprotein that is responsible for receptor binding and membrane fusion 10 In coronaviruses infection the S protein plays an im portant role in the immune responses against the virus as neutralizing antibody passive antibody protection and cellu lar immunity have all been related to this protein 11 How ever multi protein structures such as virus like particles VLPs composed of all major structural proteins that mimic the organizations and conformations of authentic native par ticles but lack the viral genome would be more likely to stimulate stronger cellular and humoral immune responses compared to a single protein vaccine containing only the S protein Indeed we have demonstrated for another animal disease bluetongue disease of sheep that while single or combined protein vaccines were protective in sheep against virulent BTV infection BTV VLPs were much better vaccines 12 13 During coronavirus assembly the N protein which is lo cated inside the virion complexed with the viral RNA 14 by Elsevier B V All rights reserved blue or subjected to Western blotting WB analysis using appropriate SARS CoV antibodies 21 2 5 Immunofluorescence and fluorescence activated cell sorter FACS analysis The Sf9 cells were grown in chamber slides and infected with the recombinant baculovirus at an MOI of 4 for 48 h immunofluorescence analysis was performed as described previously 23 and examined on a Zeiss LSH 510 microscope For FACS analysis Sf9 cells were infected as mentioned above and the analysis was performed as described previously 23 on a Becton Dickinson FACSCalibur analyser run ning CellQuest software Becton Dickinson 3 Results 3 1 Generation of recombinant baculoviruses expressing individual SARS CoV proteins inter protein interactions in the formation of capsid structures To determine whether each of the major SARS CoV pro teins could be expressed by single recombinant baculoviruses each structural gene was used to generate a recombinant baculovirus that expressed the S E M or N proteins indi vidually in Sf9 cells Selected recombinant viruses were am plified and the expression of SARS CoV protein was analyzed by SDS PAGE in Sf9 infected cells Each recombinant virus synthesized a protein of the expected size of the SARS CoV proteins S N M or E Fig 1A In addition the specificity and authenticity of each recombinant protein were confirmed by WB analysis with mouse anti SARS polyclonal antibody Fig 1B To determine if these recombinant proteins could interact with each other and assemble into particulate structures Sf9 VLPs in Sf9 infected cells Sf9 cells were infected with single re combinant baculoviruses at a MOI of 4 and analyzed by A SDS E Mortola P Roy FEBS Letters 576 2004 174 178 175 2 4 Electron microscopy EM For negative staining aliquots of the VLP samples were placed on plastic carbon coated grids and stained with 2 phosphotungstic acid pH 5 2 Immunogold labeling was performed on VLP samples as described previously 22 For thin sectioning infected cells were fixed agar embedded and cut into smaller cubes The cubes were embedded in epoxy resin and ultrathin sections were cut and mounted onto copper grids All the samples were examined on a Jeol 1200EX transmission microscope In this report we used single and multigene baculovirus expression vectors to express all four structural proteins of SARS CoV and to investigate the assembly of VLPs as a first step to the development of VLP vaccine for SARS Our studies here present clear evidence of the expression and spontaneous assembly of the three structural proteins in appropriate mo lar ratios as well as of their release from the cell surface as VLPs 2 Materials and methods 2 1 Cloning and construction of recombinant baculoviruses The plasmid genes encoding the small envelope protein E the membrane protein M the nucleocapsid protein N and the major structural spike glycoprotein S of SARS cloned into a pCR blunt TopoII vector Invitrogen Inc were obtained from Dr Kirill Kalnin Acambis Inc MA The full length M N and E genes were amplified by polymerase chain reaction PCR using primers which included recognition sites for BamHI The amplified genes were cloned into the BamHI site of the pAcYM1 vector under the control of the polyhedrin promoter The full length gene encoding the S protein was excised from pBlunt vector with an EcoRI restriction enzyme digest coupled to BglII linkers and inserted into a BglII site of the baculovirus transfer vector pTriEx 1 The correct orientation of the insertions was exam ined by PCR and restriction enzyme analysis The constructs were co transfected with triple cut linearized Autographa californica AcNPV DNA to produce recombinant baculoviruses that individually ex pressed the M N E and S proteins In order to obtain a single recombinant baculovirus that expressed the M E and S proteins two new constructs were generated The first contained the S and E genes cloned into the BamHI and BglII sites respectively of the pAcVC3 vector under the control of the polyhedrin promoter For the second construct the M gene was inserted into the BglII site under the control of the p10 promoter of a pAcP102x vector which also contains the GUS gene to allow easy selection of double recombinants when transfected with AcNPV DNA in Spodoptera frugiperda Sf9 insect cells 2 2 Purification of VLPs Assembled VLPs were isolated either from the cytoplasm of infected cells or from the culture medium Infected cells were pelleted by cen trifugation at 4000 rpm for 15 min resuspended in TEN bu er 10 mM Tris HCl pH 7 4 1 mM EDTA and 1 M NaCl with 1 Triton X 100 and dounce homogenized on ice After centrifugation 3500 rpm for 30 min the clarified supernatant was centrifuged on a linear 30 45 w w sucrose gradient in TEN bu er at 27000 rpm for 3 h The opales cent band containing the particles was clearly visible in the middle of the gradients To isolate VLPs from the culture media of the infected cells the media were clarified by centrifugation at 3500 rpm for 30 min and then centrifuged at 27000 rpm for 50 min on a 15 w w sucrose cushion in TEN bu er at 4 C176C The pelleted particles were recovered in TNE bu er 10 mM Tris HCl pH 7 4 1 mM EDTA and 0 1 M NaCl and stored for analysis Alternatively VLPs were further purified on a linear sucrose gradient 30 45 w w as above and recovered in TNE bu er to a final concentration of 1 lg ll 2 3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE and Western blotting The Sf9 cells were harvested lysed and resolved by 10 or 15 SDS PAGE 20 The gels were either stained with Coomassie brilliant PAGE followed by Coomassie blue staining and B WB analysis using mouse anti SARS polyclonal antibody The figure shows the clearly expressed SARS proteins when compared to the mock infected cells It should be noted that the M protein appears as a double band in the gel and the larger form of the protein reacted poorly with our antibody C EM analysis of negatively stained cell sections revealed the pres ence of VLPs These particles were visible in the cytoplasm after 2 days co infection with the M and E expressing recombinant baculoviruses Bar 100 nm Fig 1 Expression of S N M and E SARS proteins and detection of cells were infected with recombinant baculoviruses expressing M E or S alone or co infected in combination of M and E M and S or M E and S EM analysis of the negatively stained infected cell sections exhibited no particulate structures when each protein was expressed alone However cells infected with recombinant viruses expressing M and E had large numbers of particulate structures with a diameter of approximately 100 nm distributed throughout the cytoplasm Fig 1C In con trast no structures could be detected in the cells that were co infected with recombinant viruses expressing M and S The results demonstrate that the M and E proteins were su cient for the assembly of particles but in the absence of the E pro tein M and S could not assemble into VLPs Cells co infected with three di erent recombinant baculoviruses M E and S resulted insu cient quantity of particles assembled with three proteins to be detectable by our assay system data not shown To obtain purified VLPs containing M and E proteins Sf9 cells were co infected with two recombinant baculoviruses expressing M and E proteins 48 h post infection PI cells were lysed and VLPs were purified by sucrose gradient centrifuga tion The assembled particles were isolated and the expression of SARS CoV proteins was assessed by SDS PAGE Only two protein bands were visible one of approximately 30 kDa in size corresponding to the M protein and the other of 10 kDa purification regime and the EM staining reagents Fig 2C In the absence of S protein expression there were no spike pro jections and this gave an even smooth appearance to the spherical particles 3 2 Generation of a recombinant baculovirus expressing three SARS proteins and demonstration of SARS VLPs assembly and release In order to enhance the VLP assembly a triple recombinant baculovirus that would express S M and E proteins was generated When protein expression by this recombinant virus was assessed by SDS PAGE very high levels of expression of SARS proteins particularly S and M proteins were achieved approaching 500 lg recombinant protein per 1C210 6 infected cells Fig 3A WB analysis of the infected cell lysate using a mouse anti SARS polyclonal antibody revealed the presence of all three SARS CoV proteins Fig 3B By immunofluorescence we examined the SARS CoV S protein for expression on the surface of the Sf9 cells when infected with the triple recombinant baculovirus The S gly coprotein was observed at the periphery of the fixed infected cells demonstrating the distribution of the S protein on the plasma membrane Fig 3C Also FACS analysis using anti S monoclonal antibody clearly demonstrated the expression of the S protein on the live cell surface in comparison to the uninfected control cells data not shown To discern if assembled VLPs containing the S envelope glycoprotein were indeed released from the infected cell mimicking the coronavirus morphogenesis Sf9 cells were in 176 E Mortola P Roy FEBS Letters 576 2004 174 178 in size identical to the E protein size M protein runs slightly slower than the predicted size of 25 kDa in this analysis probably due to the glycosylation of the protein on expression in the baculovirus system The sucrose gradient purification revealed that the two SARS envelope proteins M and E could indeed self assemble into particles Fig 2A Importantly WB analysis confirmed that the two visible bands were M and E proteins which reacted with the mouse anti SARS polyclonal antibody Fig 2B To visualize the VLP morphology the VLPs were examined by negative staining and EM Smooth particles with approximate diameter of 100 nm were visualized indicating that the VLPs were stable enough to tolerate the Fig 2 Analysis of VLPs formed by Sf9 cells co infected with M and E expressing recombinant baculoviruses The VLPs were purified using a sucrose gradient and the protein composition was determined by A SDS PAGE followed by Coomassie blue staining and B WB analysis using a mouse anti SARS polyclonal antibody C EM of negatively stained smooth SARS VLPs purified by sucrose gradient Bar 100 nm fected with the triple recombinant baculovirus and were har vested at a time course of 48 72 and 96 h PI Both the cell lysates and culture medium were examined for the presence of VLPs All three proteins were visible in both cell lysates and in Fig 3 Expression of S M and E SARS proteins in Sf9 infected cells To examine protein expression levels the cells were infected with the triple recombinant baculovirus at a MOI of 4 and analyzed by A SDS PAGE followed by Coomassie blue staining and B WB analysis using mouse anti SARS polyclonal antibody C Immunofluorescence analysis of fixed cells infected with the triple recombinant baculovi ruses Expression of the SARS S glycoprotein was detected with a primary anti S monoclonal antibody and secondary anti mouse FITC conjugated antibody on the plasma membrane of infected cells Bar 10 lm the media from 48 h onwards particularly high level of S protein was present in the cells both at 48 and 72 h but not in the medium However at 96 h larger amounts of proteins were synthesized and released into the medium Fig 4 Therefore assembled VLPs released at 96 h PI were subjected to further investigation The SDS PAGE analysis of putative VLPs purified from the supernatant showed three distinct protein bands of approxi mately 180 30 and 10 kDa corresponding to the S M and E SARS CoV proteins respectively Fig 5A The expression of authentic proteins was subsequently confirmed by WB analy sis using a mouse anti S polyclonal antibody Fig 5B In order to investigate whether the N protein could be in corporated within the VLPs Sf9 cells were co infected with the triple recombinant baculovirus together with the single re combinant virus expressing the N protein Despite the fact that the N protein was expressed in the infected Sf9 cells the ex amination of the purified VLPs released into the media 96 h PI lished by the fact that the M protein is absolutely necessary in the organization of the viral envelope during assembly 7 4 Discussion In this paper we describe the production of SARS CoV like particles by expression of the viral structural proteins in the baculovirus system By co expression of the M and E proteins we were able to produce well assembled spikeless but stable VLPs These smooth surfaced particles are probably an in termediate particle in virus assembly The formation of VLPs by these two proteins has been reported for other coronavi ruses 16 19 and a very recent report has been published regarding the production of SARS VLPs by multiple co in fection with single baculoviruses expressing M E and S pro teins individually In this report the VLPs did not bud from the insect infected cells but instead they were isolated from cell lysates 24 However when we co infected Sf9 cells with three recombinant viruses expressing M E and S proteins we were not able to generate stable VLPs with the spike protein on the surface that could be isolated from culture medium For this reason and because of the ine ciency of the assembly process by co infection with several recombinant baculoviruses we generated a single recombinant baculovirus that expressed all three SARS proteins simultaneously This in turn means that every infected cell in a culture receives all of the structural proteins of the virus and expresses them all at the same time This feature is particularly important for the e cient assembly of multi protein complexes where simultaneous infection with multiple baculoviruses is essential This is the first report describing the co expression of the three SARS proteins M E and S at very high level from a E Mortola P Roy FEBS Letters 576 2004 174 178 177 still had only S M and E proteins but did not exhibit the presence of the N protein data not shown 3 3 Morphology of released VLPs and the presence of S protein on the particles We used EM analysis to examine if the morphology of VLPs that consist of S protein in addition to the M and E proteins is di erent to the VLPs formed by only the M and E proteins The negatively stained VLPs exhibited a very di er ent morphology to that of the M and E VLPs with the S protein had a spherical morphology with distinctive fine spike projections Fig 5C Clearly the three SARS CoV proteins synthesized by a triple recombinant baculovirus in insect cells had