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JOURNAL OF VIROLOGY Nov 2008 p 11318 11330 Vol 82 No 22 0022 538X 08 08 00H110010 doi 10 1128 JVI 01052 08 Copyright 2008 American Society for Microbiology All Rights Reserved The M E and N Structural Proteins of the Severe Acute Respiratory Syndrome Coronavirus Are Required for Efficient Assembly Trafficking and Release of Virus Like Particles H17188 Y L Siu 1 K T Teoh 1 J Lo 2 C M Chan 3 F Kien 1 N Escriou 4 S W Tsao 5 J M Nicholls 2 R Altmeyer 6 J S M Peiris 1 3 R Bruzzone 1 and B Nal 1 HKU Pasteur Research Centre 8 Sassoon Road Hong Kong SAR China 1 Department of Pathology The University of Hong Kong Queen Mary Hospital Hong Kong SAR China 2 Department of Microbiology The University of Hong Kong 21 Sassoon Road Hong Kong SAR China 3 Institut Pasteur Unite deGe ne tique Mole culaire des Virus Respiratoires URA CNRS 3015 25 28 Rue du Docteur Roux 75724 Paris Cedex 15 France 4 Department of Anatomy The University of Hong Kong 21 Sassoon Road Hong Kong SAR China 5 and CombinatoRx Singapore Pte Ltd 11 Biopolis Way 138667 Singapore 6 Received 20 May 2008 Accepted 7 August 2008 The production of virus like particles VLPs constitutes a relevant and safe model to study molecular determinants of virion egress The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans SARS CoV is still controversial Recent studies have shown that SARS CoV VLP formation depends on either M and E proteins or M and N proteins Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells This suggests that the mechanism of SARS CoV assembly differs from that of other studied coronaviruses which only require M and E proteins for VLP formation When coex pressed the native envelope trimeric S glycoprotein is incorporated onto VLPs Interestingly when a fluores cent protein tag is added to the C terminal end of N or S protein but not M protein the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state of the art imaging technologies Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS CoV egress Coronaviruses are positive sense RNA enveloped viruses that belong to the Coronaviridae family in the Nidovirales or der These viruses are found in a wide variety of animals and can cause respiratory and enteric disorders of diverse severity 11 18 In the past 5 years several human and animal coro naviruses have been discovered including the highly patho genic virus responsible for the severe acute respiratory syn drome SARS CoV 34 58 60 64 68 69 Coronavirus particles consist of a helical nucleocapsid structure formed by the association between nucleocapsid N phosphoproteins and the viral genomic RNA which is surrounded by a lipid bilayer where three or four types of structural proteins are inserted the spike S the membrane M and the envelope E proteins and for some coronaviruses only the hemagglu tinin esterase HE protein for a review see reference 14 Once sufficient amounts of new genomic RNA and structural proteins have been produced assembly of particles occurs Assembly and release of virions are the last stages of the virus life cycle The triple spanning membrane glycoprotein M drives the assembly of coronavirus which bud into the lumen of the endo plasmic reticulum Golgi intermediary compartment ERGIC 32 33 62 63 M is the most abundant envelope protein that sorts viral components to be incorporated into virions M oligo merization mainly driven by its transmembrane domain is believed to allow the formation of a lattice of M proteins at ERGIC membranes 16 41 S and E membrane proteins are integrated into the lattice through lateral interactions with M whereas N and viral RNA interact with M C terminal domain which is exposed to the cytosol 4 8 15 19 30 36 48 54 55 The coronavirus S protein responsible for receptor binding and membrane fusion does not seem to have any major role in coronavirus assembly Recent studies show that E is a viro porin that forms ion channels 46 66 67 Despite its minor incorporation into virion particles 7 22 40 the small E protein plays an important but not fully understood role in virus morphogenesis and budding 20 35 70 Studies per formed on coronaviruses including the SARS CoV demon strate that depletion of the E gene from coronavirus genome strongly diminish virus growth and particle formation 9 12 35 37 57 The N protein self associates and encapsidates the RNA genome for incorporation into budding virions through interactions with the M protein independently of E and S proteins 24 52 53 61 For SARS CoV the interaction of N with M was described to be independent of viral RNA 25 45 Work on mouse hepatitis virus MHV bovine coronavirus infectious bronchitis virus and transmissible gastroenteritis vi rus has established that E and M proteins are necessary and sufficient for assembly of virus like particles VLPs which share size and morphological features with real viruses 1 2 7 8 38 65 Nevertheless the minimal requirement for assembly of SARS CoV VLPs is still controversial Y Huang et al described formation of VLPs in transfected human 293 renal Corresponding author Mailing address HKU Pasteur Research Centre 8 Sassoon Road Hong Kong SAR China Phone 852 2816 8403 Fax 852 2872 5782 E mail bnal hku hk Supplemental material for this article may be found at http jvi asm org H17188 Published ahead of print on 27 August 2008 11318 on May 11 2015 by North Carolina State University Libraries http jvi asm org Downloaded from epithelial cells that only required coexpression of the M and N viral proteins 29 On the contrary other studies described that coexpressed M and E proteins were sufficient for release of sedimentable particles from transfected mammalian cells 27 or insect cells using a baculovirus expression system 26 50 A few groups have proposed immunization with SARS CoV VLPs as an effective vaccine strategy VLPs produced in insect cells or chimeric MHV SARS CoV VLPs produced in mammalian cells were used in these studies 42 44 Our objective was to delineate the molecular mechanisms that regulate SARS CoV egress in mammalian cells Here we demonstrate that whereas VLPs could hardly be recovered from culture medium of cells coexpressing combinations of M and E or M and N proteins coexpression of both SARS CoV E and N with M allowed the release of significant levels of VLPs within 1 day When coexpressed the trimeric S protein was found in VLPs Therefore in apparent contrast to other coronaviruses SARS CoV egress strongly depends on three structural proteins M E and N The addition of fluorescent tags to viral structural proteins allowed us to visualize the egress of fluorescent VLPs in living cells Monitoring of VLP egress in living cells will be a powerful new tool for studying the involvement of viral and cellular factors during the late stages of the SARS CoV life cycle MATERIALS AND METHODS Cells and culture conditions The Vero E6 African green Monkey kidney cell line was cultured in Dulbecco modified Eagle medium DMEM supplemented with 10 heat inactivated fetal calf serum 100 U of penicillin and 100 H9262gof streptomycin per ml at 37 C with 5 CO 2 Plasmid constructions cDNAs coding for the SARS CoV M E S and N structural proteins were codon optimized for mammalian cells and synthesized by GeneArt Regensburg Germany The M cDNA was amplified by PCR and subcloned into the pIRES plasmid vector BD Biosciences San Jose CA be tween the NheI and EcoR I restriction sites of the upstream multiple cloning site The E cDNA was amplified by PCR and inserted either into the pcDNA3 1 plasmid vector between the KpnI and NotI restriction sites or into the pIRES plasmid vector between the SalI and NotI restriction sites of the downstream multiple cloning site The S cDNA was amplified by PCR and inserted either into the pcDNA3 1 plasmid vector between the NheI and ApaI restriction sites The pcDNA Nflag plasmid was constructed from the original pCRScript Nflag pro duced by GeneArt Regensburg Germany using the KpnI and XhoI restriction sites The Flag tag was in fusion with the 3H11032 end of N cDNA and separated from N cDNA by six nucleotides encoding two glycine residues The cDNAs coding for the enhanced yellow eYFP cyan eCFP and green eGFP fluorescent pro teins and the monomeric red fluorescent protein mRFP1 5 were amplified by PCR from plasmids purchased from Clontech Laboratories Takara Bio Shiga Japan and subcloned into pcDNA3 1 or pIRES vectors The S N or M cDNAs were then inserted 5H11032 to the fluorescent protein cDNAs using the ClaI and ApaI sites S and N or the XhoI and MluI M restriction sites The two fused cDNAs were separated by two codons encoding glycines Antibodies The M and E proteins were detected with rabbit polyclonal anti bodies raised against the C terminal extremity of each protein The rabbit poly clonal antibody against the M C terminal domain was purchased from ProSci Inc Poway CA The rabbit polyclonal antibody against the E protein was produced by Nicolas Escriou Institut Pasteur Paris France using a C terminal peptide The Flag tag was detected with the mouse monoclonal immunoglobulin G1 M2 antibody purchased from Sigma Aldrich The mouse polyclonal antibody against the S protein was obtained by immunizing mice with purified S trimers expressed in mammalian cells as described previously 31 The mouse mono clonal antibody against the N protein was a generous gift from K H Chan Department of Microbiology University of Hong Kong and produced as de scribed previously 56 Transient transfections and production of SARS CoV VLPs A total of 8 H11003 10 5 cells were plated in 75 cm 2 dishes incubated overnight and transfected with plasmid constructs using a FuGENE 6 transfection reagent Roche Basel Swit zerland according to the manufacturer s instructions Briefly 54 H9262l of FuGENE 6 transfection reagent was mixed with DMEM followed by incubation for 5 min and then 6 H9262g of each plasmid was added The FuGENE 6 plasmid mixture was incubated for 30 min at room temperature Cell medium was discarded and replaced with 3 ml of warm DMEM The FuGENE 6 plasmid mixture was added to the cells After3hofincubation at 37 C the medium containing the trans fection mixture was discarded and 10 ml of fresh medium was added Cells were incubated for 21 or 45 h Purification of SARS CoV VLPs At 24 or 48 h posttransfection cell medium was collected and cleared by centrifugation at low speed 1 000 H11003 g for 10 min to pull down cell debris After passage through 0 45 H9262m pore size filter cleared cell medium was then loaded on top of 20 sucrose cushions and ultracentri fuged for3hat28 000 rpm using a SW41 rotor Belkman Coulter Inc Fuller ton CA VLP containing pellets were resuspended in TNE buffer 50 mM Tris HCl 100 mM NaCl 0 5 mM EDTA pH 7 4 Western blot analysis of VLPs and cell lysates For Western blot analysis of purified VLPs 15 H9262l of resuspended pellets from ultracentrifuged culture me dium was mixed with 5 H9262l of lithium dodecyl sulfate containing loading buffer and loaded onto 4 to 12 polyacrylamide gels NuPAGE Novex Bis Tris Mini Gels Invitrogen Carlsbad CA Electrophoresis was performed with the NuPAGE morpholinepropanesulfonic acid sodium dodecyl sulfate SDS run ning buffer from the same manufacturer Alternatively resuspended pellets from 48 h time points were loaded on top of 20 to 60 sucrose gradients and ultra centrifuged for 3 5 h at 26 700 rpm 27 Twenty fractions were collected and analyzed by Western blotting For Western blot analysis of cell lysates cells were washed twice with 1H11003 phosphate buffered saline PBS at 24 or 48 h posttrans fection and lysed in lysis buffer containing 1 Triton X 100 150 mM NaCl 20 mM Tris HCl pH 7 5 and 1 mM EDTA for 15 min on ice with frequent vortexing The lysates were then cleared by centrifugation at 16 100 H11003 g for 15 min at 4 C and analyzed by Western blotting Next 15 H9262l of each lysate was mixed with 5 H9262l of lithium dodecyl sulfate loading buffer and loaded on polyac rylamide gels For detection of E but not S samples were treated with dithio threitol and heated at 95 C for 5 min before migration on polyacrylamide gels To detect both E and trimers of S from same preparations samples were split and either treated or not treated before loading of two separate gels for E and S detection respectively The results for M and Nflag were similar with or without treatment Electron microscopy For transmission electron microscopy experiments transfected cells were harvested at 24 and 48 h posttransfection Cells were detached using 10 mM EDTA fixed in 2 5 glutaraldehyde in cacodylate buffer and postfixed with 1 osmium tetroxide OsO 4 in cacodylate buffer for1hat room temperature The cells were then embedded in 2 agarose to form cell blocks that were dehydrated in graded series of ethanol and embedded in epoxy resin Ultrathin sections were stained for 45 min with 2 aqueous uranyl acetate and for 30 min with Reynolds lead citrate For analysis of secreted VLPs the VLPs were purified from the cell medium by ultracentrifugation on a 20 sucrose cushion and separated on a 20 to 60 discontinuous sucrose gradient and fraction 10 was collected and then concentrated by ultracentrifugation in TNE buffer pH 7 4 for1hat28 000 rpm Next 5 H9262l of the VLP suspension mixed with an equal volume of negative stain 2 aqueous uranyl acetate and 2 phosphotungstate solution pH 7 0 was placed onto a Formvar carbon coated copper grid for 2 min and the excess suspension was drained The grids were viewed and photographed with a Philip CM100 electron microscope at 80 kV Fluorescence microscopy For fluorescence microscopy on fixed cells Vero E6 cells were grown on glass coverslips transfected and analyzed at 24 h posttrans fection Cells were washed with PBS nuclei were stained with DAPI 4H11032 6H11032 diamidino 2 phenylindole and coverslips were mounted on glass slides for anal ysis Fixed cells were visualized under an AxioObserver Z1 inverted motorized fluorescence microscope using the ApoTome module and piloted through the Axiovision 4 6 software and images were acquired through an MRm AxioCam high resolution charge coupled device camera Carl Zeiss Germany For fluo rescence microscopy on live cells Vero E6 cells were grown in a glass bottom culture dish MatTech transfected and analyzed at 24 h posttransfection The cells were washed and the medium was changed to Hanks balanced salt solution OptiMEM prewarmed culture medium Wide field image acquisitions of live cells were obtained using the system described above Confocal acquisitions of live cells were acquired at the Hong Kong University Core Imaging Facility using an AxioObserver Z1 inverted motorized fluorescence microscope equipped with a spinning disc confocal imaging system UltraVIEW ERS Perkin Elmer Shel ton CT For brefeldin A BFA treatment cells were incubated with 6 H9262g per ml of BFA for the indicated times To release the BFA effect the cells were washed three times in 1H11003 PBS and reincubated in normal medium for the indicated times VOL 82 2008 M E AND N STRUCTURAL PROTEINS OF SARS 11319 on May 11 2015 by North Carolina State University Libraries http jvi asm org Downloaded from RESULTS Efficient production and release of SARS CoV VLPs are driven by coexpression of M N and E structural proteins To determine the optimal conditions for efficient SARS CoV VLP production we chose to coexpress different combinations of structural proteins in the Vero E6 African green monkey cell line which is permissive to SARS CoV replication 34 cDNAs encoding SARS CoV structural proteins M E and N were codon optimized for expression in mammalian cells Un derstanding the importance of maintaining a low E M ratio in transfected cells to ensure low incorporation of E into VLPs and to prevent the potential formation of E containing vesi cles we reasoned that the use of the pIRES bicistronic plasmid should be appropriate This plasmid presents the double ad vantage to ensure coexpression of both cDNAs in transfected cell and allows a reduced rate of translation of the downstream gene by utilizing a partially disabled internal ribosome entry site IRES sequence We therefore subcloned M upstream of E cDNA in the pIRES vector N was expressed from a pcDNA plasmid with a C terminal Flag tag Vero E6 cells were either not transfected or transfected with pcDNA E pIRES M or pIRES M E alone pIRES M plus pcDNA Nflag and pIRES M E plus pcDNA Nflag Fig 1A At 24 and 48 h posttrans fection the culture medium and cells were harvested The culture medium was subjected to ultracentrifugation on a 20 sucrose cushion to isolate VLPs and the SARS CoV structural proteins assembled into VLPs were analyzed by Western blot ting The M protein was readily detected at both 24 and 48 h posttransfection in lysates from Vero E6 cells transfected with pIRES M pIRES M E pIRES M plus pcDNA Nflag and pIRES M E plus pcDNA Nflag Fig 1A upper panels As described previously three major forms of M which corre spond to different glycoforms were detected by SDS polyacryl amide gel electrophoresis two bands at ca 18 and 28 kDa and a smear at higher molecular sizes reflecting heterogeneity of glycosylation patterns 51 As expected higher levels of M were detected in cell lysates at 48 h than at 24 h posttransfec tion Whereas a 10 kDa protein corresponding to E could be easily detected in cell lysates from cells transfected with pcDNA E plasmid its expression was much lower in cells transfected with the pIRES M E bicistronic vector The Nflag protein was found in cell lysates from both pIRES M plus pcDNA Nflag and pIRES M E plus pcDNA Nflag transfected cells at similar levels A major band corresponding to a protein with an apparent molecular size of 45 kDa was detected Strikingly the efficacy of VLP production was dramatically affected by the combination of viral proteins coexpressed Fig 1A lower panels Although M was not detected in ultracen trifuged culture medium from pIRES M pIRES M E or pIRES M plus pcDNA Nflag transfected cells at 24 h post transfection significant levels were found in preparations from pIRES M E plus pcDNA Nflag transfected cells Similarly the N protein was only detected in ultracentrifuged culture me dium from pIRES M E plus pcDNA Nflag transfected cells at this early time point Signals for E were below the limit of detection at 24 h and detectable only at 48 h Finding low amounts of E in VLPs is in accordance with the minor pres ence of this protein in coronavirus particles despite its impor tant role for virion assembly and budding 9 20 37 57 65 In conclusion only when M E and N proteins were coexpressed VLPs that contained the M and N proteins could be isolated from the culture medium at 24 h posttransfection At 48 h posttransfection E was found in ultracentrifuged culture medium from cells transfected with the pcDNA E plas mid alone This is in agreement with previously published data that describe secretion of E proteins independently of other viral elements 7 47 50 At this time point trace amounts of M were detected in ultracentrifuged culture medium from pIRES M and pIRES M E transfected cells whereas signifi cant levels of M and N were found in pIRES M plus pcDNA Nflag ultracentrifuged culture medium Independent secretion of SARS CoV M proteins from Vero E6 cells as well as pro duction of M E VLPs was described by Mortola and Roy 50 at 4 days posttransfection The production of SARS CoV M N VLPs has already been reported by Y Huang et al in trans fected 293 renal epithelial cells at 63 h posttransfection 29 Very interestingly in the conditions described here signifi cantly higher levels of M and N proteins were found in purified VLPs from pIRES M E plus pcDNA Nflag than in those from pIRES M plus pcDNA Nflag transfec