JOURNAL OF VIROLOGY Dec 2008 p 12392 12405 Vol 82 No 24 0022 538X 08 08 00H110010 doi 10 1128 JVI 01219 08 Copyright 2008 American Society for Microbiology All Rights Reserved Topology and Membrane Anchoring of the Coronavirus Replication Complex Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning H17188 Monique Oostra Marne C Hagemeijer Michiel van Gent Cornelis P J Bekker Eddie G te Lintelo Peter J M Rottier and Cornelis A M de Haan Virology Division Department of Infectious Diseases all other cleavages are performed by the 3C like main proteinase located in nsp5 2 11 17 55 64 pp1a which contains the first 11 nsps including the proteinases is three to five times more abundantly produced than pp1ab which additionally gives rise to nsp12 to nsp16 13 Functions involving the actual replication and tran scription of the viral genome have been assigned to several of the orf1b encoded nsps like RNA dependent RNA polymerase Corresponding author Mailing address Virology Division Depart ment of Infectious Diseases for the reverse primers used see Table S1 in the supplemental material For nsp3 the PCRs were performed on the constructs with the mutated N glycosylation sites The PCR products were cloned into the pGEM T Easy vector Promega and the sequences were con firmed by sequence analysis Fragments were obtained by digestion with EcoRI and BamHI and cloned into the EcoRI BamHI digested pTug EGFP glyc vector By using combinations of the primers used to create the C terminal deletions and primers for N terminal deletions the sequences encoding each of the hy drophobic domains of MHV nsp6 were also amplified separately These se quences were cloned into the pGEM T Easy vector Promega and confirmed by sequence analysis Fragments were obtained by digestion with EcoRV and BamHI and cloned into the EcoRV BamHI digested pTugM N nsp6 m EGFP glyc plasmid thereby creating constructs encoding the MHV nsp6 fragments fused N terminally to the MHV M tag and C terminally to the EGFP tag containing the N glycosylation site The construct containing SARS CoV nsp4 in which the N glycosylation site had been removed by mutation has been described previously 37 This con struct was used to create C terminal deletion mutants lacking one to three of the hydrophobic domains PCRs were performed with primer 3648 and primer 3848 3650 or 3651 the products were cloned into the pGEM T Easy vector Pro mega and the sequences were confirmed by sequence analysis Fragments were obtained by digestion with EcoRI and BamHI and cloned into the EcoRI BamHI digested pTug EGFP glyc vector The construct containing MHV nsp4 has also been described previously 37 The glycosylation sites in MHV nsp4 were mutated by performing sequential site directed mutagenesis reactions with primers 3758 and 3759 and primers 3760 and 3761 The construct with the mutated glycosylation sites was used to create C terminal deletion mutants by performing PCRs with primer 2890 and primer 3847 3756 or 3757 The PCR products were cloned into the pGEM T Easy vector Promega and the sequences were confirmed by sequence analysis Fragments were obtained by digestion with EcoRI and BamHI and cloned into the EcoRI BamHI digested pTug EGFP glyc vector The constructs encoding the equine arterivirus EAV membrane protein N terminally extended with the M N tag EAV M H11001 9A 10 and the 8a protein C terminally tagged with the EGFP tag with or without the N glycosylation site 36 have been described previously Infection and transfection Subconfluent monolayers of OST7 1 cells grown in 10 cm 2 tissue culture dishes were inoculated with vTF7 3 at a multiplicity of infection of 10 for 1 h after which the medium was replaced with a transfection mixture consisting of 0 5 ml of DMEM without FCS but containing 10 H9262l Lipofectin Invitrogen and 5 H9262g of each selected construct After a 5 min incubation at room temperature 0 5 ml of DMEM was added and incubation was continued at 37 C At 3 h postinfection p i the medium was replaced with culture medium Where indicated tunicamycin 5 H9262g ml or brefeldin A 6 H9262g ml was added to the culture medium at 3 h p i Metabolic labeling and immunoprecipitation Prior to labeling the cells were starved for 30 min in cysteine and methionine free modified Eagle s medium containing 10 mM HEPES pH 7 2 and 5 dialyzed FCS This medium was replaced with 1 ml of a similar medium containing 100 H9262Ci of 35 S in vitro cell labeling mixture Amersham after which the cells were further incubated for the indicated time periods After labeling the cells were washed once with phosphate buffered saline PBS containing 50 mM Ca 2H11001 and 50 mM Mg 2H11001 and then lysed on ice in 1 ml of lysis buffer 0 5 mM Tris pH 7 3 1 mM EDTA 0 1 M NaCl 1 Triton X 100 per 10 cm 2 dish The lysates were cleared by cen trifugation for 5 min at 15 000 rpm and 4 C Coupled in vitro transcription and translation reactions were performed with the TNT coupled reticulocyte lysate system from Promega according to manufacturer s instructions in the presence of 35 S in vitro labeling mixture Amersham but without the use of microsomal membranes Radioimmunoprecipitations were performed essentially as described previ ously 35 200 H9262l aliquots of the cell lysates or 5 H9262l volumes of in vitro trans lation reaction mixtures were diluted in 1 ml detergent buffer 50 mM Tris pH 8 0 62 5 mM EDTA 1 NP 40 0 4 sodium deoxycholate 0 1 sodium dodecyl sulfate SDS containing antibodies 2 H9262l rabbit anti EGFP or rabbit anti HA serum or 25 H9262l of J1 3 monoclonal anti MHV M serum The immu noprecipitation mixtures were incubated overnight at 4 C The immune com plexes were adsorbed to Pansorbin cells Calbiochem for 60 min at 4 C and subsequently collected by centrifugation The pellets were washed three times by resuspension and centrifugation with RIPA buffer 10 mM Tris pH 7 4 150 mM NaCl 0 1 SDS 1 NP 40 1 sodium deoxycholate The final pellets were suspended in Laemmli sample buffer LSB and heated at 95 C for 1 min before analysis by SDS polyacrylamide gel electrophoresis PAGE with 10 to 15 polyacrylamide gels Where indicated immunoprecipitates were treated with peptide N glycosi dase F PNGaseF New England BioLabs To this end the final immunopre cipitation pellets were suspended in PBS instead of LSB 2 H9262l PNGaseF was 12394 OOSTRA ET AL J VIROL on May 30 2015 by guest http jvi asm org Downloaded from added and the samples were incubated at 37 C for 2 h Before analysis by SDS PAGE 0 5 volume of a three times concentrated solution of LSB was added to the samples which were then heated at 95 C for 1 min Immunofluorescence microscopy OST7 1 cells grown on glass coverslips were fixed at the indicated times postinfection with 3 paraformaldehyde for1hatroom temperature The fixed cells were washed with PBS and permeabilized with either 0 1 Triton X 100 for 10 min at room temperature or 0 5 H9262g ml digitonin diluted in 0 3 M sucrose 25 mM MgCl 2H11001 0 1 M KCl 1 mM EDTA 10 mM PIPES pH 6 8 for 5 min at 4 C Next the permeabilized cells were washed with PBS and incubated for 15 min in blocking buffer PBS 10 normal goat serum followed by a 45 min incubation with antibodies directed against HA or against the C or N terminal domain of MHV M After four washes with PBS the cells were incu bated for 45 min with either fluorescein isothiocyanate conjugated goat anti rabbit immunoglobulin G antibodies ICN or Cy5 conjugated donkey anti mouse immu noglobulin G antibodies Jackson Laboratories After four washes with PBS the samples were mounted on glass slides in FluorSave Calbiochem The samples were examined with a confocal fluorescence microscope Leica TCS SP2 RESULTS Processing of nsp3 and nsp6 We started our studies of nsp3 and nsp6 by analyzing the co and posttranslational modifica tions of the two proteins from both SARS CoV and MHV To this end gene fragments encoding nsp3 and nsp6 of both viruses were cloned into the pTUG31 vector behind a T7 promoter and fused to an EGFP tag since no antibodies to the proteins themselves were available As the full length nsp3 encoding genome segments are very large approximately 6 kb and difficult to clone only the 3H11032 terminal 2 kb fragments which encode all of the hydrophobic domains were cloned nsp3 of both SARS CoV and MHV contains potential N gly cosylation sites NXS T in front of the first hydrophobic do main between the first and second hydrophobic domains and behind the third hydrophobic domain Fig 1 shows the local ization of the hydrophobic domains The glycosylation sites between the first and second hydrophobic domains two for SARS CoV and one for MHV have previously been shown to be functional 22 28 No potential N glycosylation sites were identified in the sequence of MHV nsp6 while for SARS CoV nsp6 an N glycosylation site was predicted between the fifth and sixth hydrophobic domains We studied the expression and processing of the nsps by in vitro translation and by using the recombinant vaccinia virus bacteriophage T7 RNA polymerase vTF7 3 expression sys tem OST7 1 cells were infected with vTF7 3 transfected with plasmids containing the nsp3 EGFP or nsp6 EGFP gene and labeled with 35 S methionine from 5 to 6 h p i The cells were lysed and processed for immunoprecipitation with a rabbit polyclonal antiserum directed to the EGFP tag In parallel in vitro translations were performed with the TNT coupled re ticulocyte lysate system from Promega in the absence of mem branes to analyze the electrophoretic mobility of the nonproc essed proteins To demonstrate the presence of the N linked sugars on the nsps the proteins were expressed in the presence and absence of tunicamycin which is an inhibitor of N linked glycosylation and or the N linked glycans were enzymatically removed with PNGaseF As shown in Fig 2A the electrophoretic mobility of nsp3 expressed in OST7 1 cells in the presence of tunicamycin was similar to that of the in vitro translated product whereas the protein expressed in the absence of tunicamycin migrated slower Treatment of this latter protein with PNGaseF shifted its electrophoretic mobility to that of the in vitro translated product and of the protein expressed in the presence of tuni camycin This result confirms the addition of N linked glycans to nsp3 as has been demonstrated previously 22 28 Next the N glycosylation sites between the first and second hydro phobic domains were mutated and these proteins in fusion with EGFP nsp3 H11002glyc EGFP were also expressed in the presence and absence of tunicamycin and or treated with FIG 2 Processing of SARS CoV and MHV nsp3 and nsp6 vTF7 3 infected OST7 1 cells were transfected with the indicated constructs The cells were labeled with 35 S methionine from 5 to 6 h p i lysed and processed for immunoprecipitation with antiserum directed to the EGFP tag followed by SDS PAGE A Cells were transfected with SARS CoV or MHV nsp3 EGFP nsp3 s EGFP or nsp3 m EGFP respectively encoding constructs without or with mutation H11002glyc of the N glycosylation sites in the presence TM or absence H11002 and P of tunicamycin The constructs with intact glycosylation sites were also transcribed and translated in vitro with the TNT coupled reticulocyte lysate system from Promega ivt After immunoprecipitations the samples were mock TM H11002 or PNGaseF P treated B Cells were transfected with SARS CoV or MHV nsp6 EGFP nsp6 s EGFP and nsp6 m EGFP respectively encoding constructs in the presence H11001 or absence H11002 of tunicamycin TM The same constructs were also transcribed and translated in vitro with the TNT coupled reticulocyte lysate system from Promega ivt The positions and masses in kilodaltons of protein size markers are indicated at the left Only the relevant portion of the gels is shown VOL 82 2008 MEMBRANE ANCHORING OF CORONAVIRUS REPLICATION COMPLEX 12395 on May 30 2015 by guest http jvi asm org Downloaded from PNGaseF The presence of tunicamycin or treatment with PNGaseF did not influence the electrophoretic mobility of these mutant proteins Fig 2A This clearly demonstrates that the N glycosylation sites between the first and second hydro phobic domains are the only N glycan attachment sites The other potential sites are likely inaccessible probably because they are located on the cytoplasmic side of the membrane vTF7 3 expressed nsp6 of both viruses comigrated in the gel with the corresponding in vitro translated products both in the presence and in the absence of tunicamycin Fig 2B Also some lower molecular weight products were observed after the in vitro translation of MHV nsp6 which probably resulted from translation initiation at more downstream start codons Apparently nsp6 of SARS CoV or MHV is not N glycosylated indicating that the putative glycosylation site in the region between the fifth and sixth hydrophobic domains of SARS CoV nsp6 is either located on the cytoplasmic side of the membrane or not accessible for glycosylation for other reasons Furthermore it appeared that both nsp6 fusion proteins mi grated with lower mobility in the gel than predicted on the basis of their amino acid sequences Similar results have been obtained before for other highly hydrophobic proteins 37 41 Membrane topology of nsp3 and nsp6 In order to elucidate the membrane topology of SARS CoV and MHV nsp3 and nsp6 we studied the disposition of their amino and carboxy termini Therefore the proteins were C or N terminally ex tended with tags containing potential glycosylation sites The C termini of the proteins were fused to an EGFP tag in which an N glycosylation site had been created EGFP glyc As a control we used a fusion protein generated earlier which essentially consists of a cleavable signal sequence fused to the same tag 8a EGFP 36 For nsp3 the tag was fused to either the SARS CoV or the MHV gene fragment in which the N glyco sylation sites between the first and second hydrophobic do mains had been disabled by mutation nsp3 H11002glyc to allow dis crimination between glycosylation of the tag and that of nsp3 itself The fusion proteins were expressed with the vTF7 3 expression system in the presence and absence of tunicamycin N glycosylation of the C terminal EGFP tag would demon strate that the carboxy terminus of the protein is located on the lumenal side of the membrane For each of the proteins nsp3 or nsp6 a similar electro phoretic mobility was observed regardless of the EGFP tag used i e with or without the N glycosylation site or the presence of tunicamycin Fig 3A The control protein 8a behaved as expected In the presence of tunicamycin the pro tein with the EGFP glyc tag migrated with the same mobility as the protein with the wild type tag in the absence of tunicamy cin whereas in the absence of tunicamycin the protein with the EGFP glyc tag migrated slower Fig 3A The results demon strate that the carboxy termini of nsp3 and nsp6 of both SARS CoV and MHV are located on the cytoplasmic side of the membrane To examine the disposition of the nsp3 and nsp6 amino termini the N terminal 10 residue sequence of the MHV M protein M N which contains a well defined O glycosylation motif was fused to the amino terminus of each nsp The functionality of this tag was previously demonstrated after fu sion to the EAV type III M protein resulting in EAV M H11001 9A 10 This protein has a Nexo Cendo topology and is retained in the ER Yet it became O glycosylated upon the addition of brefeldin A a drug which causes the redistribution of Golgi enzymes including the ones involved in O glycosylation to the ER By a similar approach the location of the N termini of nsp3 and nsp6 was assessed The nsp fusion proteins contain ing the N terminal M N and a C terminal HA tag were ex pressed with the vTF7 3 expression system in OST7 1 cells in the presence or absence of brefeldin A and or tunicamycin FIG 3 Glycosylation of tagged SARS CoV and MHV nsp3 and nsp6 vTF7 3 infected OST7 1 cells were transfected with the indicated constructs The cells were labeled with 35 S methionine from 5 to 6 h p i lysed and processed for immunoprecipitation followed by SDS PAGE A Cells were transfected with constructs encoding SARS CoV or MHV nsp3 or nsp6 or encoding the SARS CoV orf8a protein 8a each fused either to a wild type EGFP tag wt or to an EGFP tag with an N glycosylation site H11001glyc The proteins were expressed in the presence H11001 or absence H11002 of tunicamycin TM Immunoprecipitations were performed with rabbit antiserum directed to the EGFP tag B Cells were transfected with constructs encoding SARS CoV or MHV nsp3 or nsp6 with a C terminal HA tag and an N terminal M N tag or with a construct encoding the EAV M protein with the same M N tag EAV M H11001 9A MHV nsp6 lacking the first hydrophobic domain nsp6 m H9004HD1H11032 contains the N terminal M N tag in combination with a C terminal EGFP tag The cells were incubated in the presence H11001 or absence H11002 of brefeldin A bref A and or tunicamycin TM Immunoprecipitations were performed with rabbit antiserum directed to the HA tag or for EAV M with monoclonal antibody J1 3 directed against the M N tag Only the relevant portions of the gels are shown 12396 OOSTRA ET AL J VIROL on May 30 2015 by guest http jvi asm org Downloaded from Tunicamycin was added to the cells expressing the nsp3 fusion protein to prevent its N glycosylation as this could obscure the detection of its O glycosylation As shown in Fig 3B the EAV M H11001 9A control protein showed a shift in electrophoretic mobility when it was ex pressed in the presence of brefeldin A In contrast the elec trophoretic mobilities of the nsp3 and nsp6 fusion proteins were unaffected by the addition of brefeldin A As expected the presence of tunicamycin did prevent the addition of N linked sugars to nsp3 These results indicate that the amino termini of SARS CoV and MHV nsp3 and nsp6 are not ac cessible to enzymes that mediate the addition of O linked sugars which is most likely explained by the cytoplasmic ex posure of these termini although misfolding of the amino terminal tag as the cause can also not be ruled out completely We do not consider the latter explanation very likely since the presence of two proline residues in the tag has previously been demonstrated to induce a conformation favorable for glycosy lation 10 Indeed when the first hydrophobic domain of MHV nsp6 was removed the resulting protein carrying the amino terminal tag now in combination with a C terminal EGFP tag did become modified by O linked sugars in the presence of brefeldin A as shown by the appearance of an extra band which runs at a slightly higher position in the gel The difference in electrophoretic mobility between the glyco sylated and unglycosylated protein species is smaller for the nsp6 mutant than for the EAV M protein because of the much higher molecular weight of EGFP tagged nsp6 Fig 3B The localization of the amino and carboxy termini of each nsp was also determined by immunofluorescence assays In these experiments nsps were used that were tagged at both ends containing the N terminal M N and a C terminal HA extension OST7 1 cells were infected with vTF7 3 and trans fected with plasmids encoding the fusion proteins The cells were fixed at 6 h p i with 3 paraformaldehyde and perme abilized under strictly controlled conditions with either Triton X 100 which permeabilizes all cellular membranes or digito nin which selectively permeabilizes the plasma membrane The type III MHV M protein which has a known Nexo Cendo topology and localizes to the Golgi compartment was used as a control A rabbit polyclonal antibody directed to the C ter minus and a mouse monoclonal antibody directed to the N terminus were used to detect this protein after the use of each of the permeabilization methods As expected the antibody directed to the C terminus detected the protein after Triton X 100 permeabilization as well as after digitonin permeabili zation whereas the antibody directed to the N te