ORIGINAL PAPERInteractions between M protein and other structural proteinsof severe, acute respiratory syndrome-associated coronavirusYi-Ching Hsieh Hui-Chun Li Shih-Chi Chen Shih-Yen LoReceived: 2 May 2008/Accepted: 21 August 2008/Published online: 16 September 2008? National Science Council Taipei 2008AbstractSevere acute respiratory syndrome-associatedcoronavirus (SARS-CoV) structural proteins (S, E, M, andNC) localize in different subcellular positions whenexpressed individually. However, SARS-CoV M protein isco-localized almost entirely with S, E, or NC protein whenco-expressed in the cells. On the other hand, only partialco-localization was observed when S and E, S and NC, or Eand NC were co-expressed in the cells. Interactionsbetween SARS-CoV M and other structural proteins butnot interactions between S and E, S and NC, or E and NCwere further demonstrated by co-immunoprecipitationassay. These results indicate that SARS-CoV M protein,similar to the M proteins of other coronaviruses, plays apivotal role in virus assembly. The cytoplasmic C-terminusdomain of SARS-CoV M protein was responsible forbinding to NC protein. Multiple regions of M proteininteracted with E and S proteins. A model for the inter-actions between SARS-CoV M protein and other structuralproteins is proposed. This study helps us better understandprotein-proteininteractionsduringviralassemblyofSARS-CoV.KeywordsSARS-CoV ? Membrane protein ?Structural proteins ? Co-localization ?Co-immunoprecipitationIntroductionSevere acute respiratory syndrome (SARS), a new infec-tious disease typically associated with fever, shortness ofbreath, cough, and pneumonia, first emerged in southernChina in November 2002. Within months of the outbreak,SARS had spread globally, affecting over 8,000 patients in29 countries with 774 fatalities 1. The etiology of SARSis associated with a newly discovered coronavirus, SARS-associated coronavirus (SARS-CoV) 24. SARS-CoVinfects many organs, including lungs, liver, and immunecells 5, 6. Subsequent studies have indicated that theSARS-CoV is of animal origin 7, and its precursor is stillpresent in animal populations within the region. Althoughthe global outbreak of SARS has been contained, there areserious concerns over its re-emergence. To date, no specifictreatment exists for this disease. Thus, further basic andclinical research is required to control the disease.SARS-CoV is phylogenetically distinct, and only dis-tantly related to the other coronavirus clades 8, 9.Coronaviruses are exceptionally large RNA viruses andemploy complex regulatory mechanisms to express theirgenomes 10. The genome structure, gene expressionpattern and protein profiles of SARS-CoV are similar tothose of other coronaviruses. Nine SARS-CoV specificmRNAs were synthesized in virus-infected cells 11.These RNA were predicted to encode 4 structural proteinsElectronic supplementary materialThe online version of thisarticle (doi:10.1007/s11373-008-9278-3) contains supplementarymaterial, which is available to authorized users.Y.-C. Hsieh ? H.-C. Li ? S.-C. Chen ? S.-Y. Lo (&)Graduate Institute of Molecular and Cellular Biology, Tzu ChiUniversity, 701, Section 3, Chung Yang Road, Hualien, Taiwane-mail: losylomail.tcu.edu.twH.-C. Li ? S.-Y. LoGraduate Institute of Medical Sciences, Tzu Chi University,Hualien, TaiwanS.-Y. LoDepartment of Laboratory Medicine and Biotechnology, Tzu ChiUniversity, Hualien, Taiwan123J Biomed Sci (2008) 15:707717DOI 10.1007/s11373-008-9278-3(spike, envelope, membrane, and nucleocapsid proteins),16 non-structural proteins, and 8 accessory proteins. Pre-vious studies on various coronaviruses indicated that thefour structural proteins (S, E, M, and NC) play roles invirion morphogenesis 12, 13. NC binds to viral RNA toform the nucleocapsid. Co-expression of M and E proteinstogether can form virus-like particles 14, 15. Interactionsbetween the M and E proteins and nucleocapsids result invirusbuddingthroughthecellularmembrane16.Through the interaction with M protein, S protein isincorporated into the viral envelope 17, 18 and the maturevirions are released from the cells. These studies suggestthat coronavirus M protein 19, 20 plays a crucial role inthe assembly of virus particles. Like other coronaviruses,SARS-CoV assembles at and buds into the lumen of theendoplasmic reticulumGolgi intermediate compartment21. Accumulation of the viral envelope proteins at thiscompartment is a prerequisite for virus assembly 22.Immuno-EM (electron microscopy) revealed that buddingoccurred at membranes of the ERGIC and the Golgi regionFig. 1 Differential subcellular localizations of SARS-CoV structuralproteins S, E, M and NC) when they were expressed individually.Cells were transfected with the plasmid expressing M-V5, the plasmidexpressing myc-E protein, the plasmid expressing NC protein, or theplasmid expressing S protein. After transfection, cells were fixed andstained with mouse anti-V5, mouse anti-myc, rabbit anti-NC, rabbitanti-S antibodies. Green color, M, NC, S, or E protein staining; bluecolor, DAPI stainingFig. 2 (a) Co-localization of SARS-CoV M protein with S, E, or NCprotein when co-expressed in Vero E6 cells. Cells were co-transfectedwith plasmids expressing the M-V5 and NC (upper), S (middle), ormyc-E (lower) proteins. After transfection, cells were fixed andstained with rabbit anti-NC and mouse anti-V5 antibodies (upper), orrabbit anti-S and mouse anti-V5 antibodies (middle), or mouse anti-myc and goat anti-mouse conjugated with Cy3 followed by anti-V5-FITC antibody (lower). Green color, M protein staining; red color,NC (or S, or E) protein staining; blue color, DAPI staining; yellowcolor, co-localization of M and other structural proteins. (b) Partialco-localization of E and NC, S and NC, or E and S proteins when theywere co-expressed in Vero E6 cells. Cells were co-transfected withplasmids expressing myc-E and NC proteins (upper); S and myc-NCproteins (middle), and myc-E and S proteins (lower). After transfec-tion, cells were fixed and stained with rabbit anti-NC (or anti-S) andmouse anti-myc antibodiesc708J Biomed Sci (2008) 15:707717123as early as 3 h post infection, demonstrating that SARS-CoV replicated surprisingly fast. Previous data also sug-gests that SARS-CoV established replication complexes atER-derived membranes 21. Later on, viral nucleocapsidswere transported to the budding sites in the Golgi regionwhere the viral glycoproteins accumulate and particleformation occurs. Assembly of SARS-CoV RNA packag-ingsignalintovirus-likeparticlesisnucleocapsiddependent 23. In this report, the protein-protein interac-tions among SARS-CoV structural proteins were studiedusing confocal microscopy and immunoprecipitation fol-lowed by Western blotting analysis. Results from this studyindicate that, similar to the M protein of other coronavi-ruses, SARS-CoV M protein plays a crucial role in theinteractions between SARS-CoV structural proteins.Materials and methodsPlasmid constructionThe construction of plasmids expressing full-length spikeand nucleocapsid proteins or encoding full-length anddeletion mutants of membrane protein plus a V5 tag wasFig. 3 (a) SARS-CoV M interaction with E protein. Vero E6 cellswere transfected with vector alone (lanes 1 and 5), plasmid encodingM-V5 (lanes 2 and 6), plasmid encoding myc-E (lanes 3 and 7), or co-transfected with both plasmids (lanes 4 and 8). Cell lysates weredirectly analyzed by Western blotting (lanes 14) or immunoprecip-itated with the anti-myc antibody prior to Western blotting (lanes 58). (b) SARS-CoV M interaction with S proteins. Vero E6 cells weretransfected with vector alone (lanes 1 and 5), plasmid encoding M-V5(lanes 2 and 6), plasmid encoding myc-S (lanes 3 and 7), or co-transfected with both plasmids (lanes 4 and 8). Cell lysates weredirectly analyzed by Western blotting (lanes 14) or immunoprecip-itated with the anti-myc antibody prior to Western blotting (lanes 58). (c) SARS-CoV M interaction with NC proteins. Vero E6 cellswere transfected with vector alone (lanes 1 and 5), plasmid encodingM-V5 (lanes 2 and 6), plasmid encoding myc-NC (lanes 3 and 7), orco-transfected with both plasmids (lanes 4 and 8). Cell lysates weredirectly analyzed by Western blotting (lanes 14) or immunoprecip-itated with the anti-myc antibody prior to Western blotting (lanes 58)J Biomed Sci (2008) 15:707717709123described previously 24, 25. Similar strategies wereperformed to prepare the plasmids expressing full-lengthspike, envelope, and nucleocapsid proteins with differenttags using primers listed in Supplementary Table I. VectorpcDNA3.1/V5-His A (Invitrogen, CA, USA) was used toadd a V5-His tag at the C-terminus of the expressed proteinwhile vector pcDNA3-cMyc tag 26 was used to add amyc tag at the N-terminus of the expressed protein.Alltheexpressionplasmidswereverifiedbysequencing.710J Biomed Sci (2008) 15:707717123Protein expression in Vero E6 cellsVero E6 cells were maintained in RPMI 1640 mediumcontaining 10% fetal calf serum, 1% glutamine (200 mM,Biological Industries, Israel), and 100 lg/ml of penicillin/streptomycin(GibcoBRL,USA).Thecells(2.52.7 9 105) were plated in the 35-mm dish. After an over-night incubation, cells were infected with a recombinantvaccinia virus carrying the T7 phage RNA polymerasegene 27. Two hours after infection, the cells were trans-fected with 0.4 lg of plasmid DNA using Effectenetransfection reagent (Qiagen, Germany). After 21 h oftransfection, the recombinant proteins in the cells wereanalyzed.Immunoprecipitation assayThe Vero E6 cells (1 9 106) were harvested 21 h aftertransfection and lysed in RIPA buffer (150 mM NaCl, 1%NP40, 0.5% deoxychloic acid, 0.1% SDS, 50 mM Tris, pH7.5). After full-speed centrifugation for 5 min in a micro-centrifuge, the supernatant was incubated with mouse anti-V5 monoclonal antibody (Invitrogen) or mouse anti-Hismonoclonal antibody (Santa Cruz Biotechnology, CA,USA) or mouse anti-myc monoclonal antibody (Oncogene,MA, USA) at 4?C overnight with shaking. The antigen-antibody complex was separated with pansorbin (Merck,Germany). The immunoprecipitated pellet was boiled for10 min in sampling buffer and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Westernblotting. In each experiment, 10% of cell lysates were usedfor expression analysis (by Western blotting assay directly)while 90% of cell lysates were used for the co-immuno-precipitation assay 25.Western blotting analysisFor Western blotting analysis, cells were dissolved insample preparation buffer after washing with PBS twice.SARS-CoV M protein is not detected in SDS-PAGE afterregular boiling treatment 24. Therefore, treatments atroom temperature were used for antigen preparation(sample buffer containing 50 mM of TrisHCl (pH 6.8),100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue,and 10% glycerol, without boiling) to detect the expressionof SARS-CoV M protein. A 4.5% acrylamide stacking geland 12% separating gel were used in this study. Whenproteins with smaller size were analyzed (e.g., membraneprotein deletion mutants), a 15% separating gel was used.After SDS-PAGE, the gel was transferred to PVDF paper(Pall Corporation, NY, USA). All procedures were carriedout at room temperature, according to previously publishedprocedures 26, 28, 29, except that the first antibody usedin this assay was mouse anti-V5 monoclonal antibody(Invitrogen) or mouse anti-His monoclonal antibody (SantaCruz Biotechnology) or mouse anti-myc monoclonal anti-body (Oncogene).Confocal microscopyAbout 2.5 9 105cells were seeded into 35-mm culturedishes. After overnight incubation at 37?C, the cells weretransfected with 0.4 lg of plasmid using the Effectenetransfection kit (Qiagen, Germany). After transfection for48 h, recombinant proteins in the cells were analyzed.Cells were fixed by acetone/methanol (1:1) at 0?C for10 min. Fixed cells were washed with incubation buffer(0.05% NaN3, 0.02% saponin, 1% skim milk in PBS) twicefor 5 min each time, then incubated with primary antibody(e.g., mouse anti-V5 monoclonal antibody, Invitrogen),which was diluted 200 fold, at 37?C for 30 min. Sampleswere washed with PBS three times (5 min each time atFig. 4 Immunoprecipitation and Western blotting analyses of SARS-CoV M protein cytoplasmic C-terminus domain interaction with NCprotein. (a) Vero E6 cells were transfected with vector alone (lanes 1and 5), plasmid encoding cytoplasmic C-terminus domain of Mprotein (i.e. M protein without its first 100 amino acids) with a V5 tag(lanes 2 and 6), plasmid encoding myc-NC (lanes 3 and 7), or co-transfected with both plasmids (lanes 4 and 8). Cell lysates weredirectly analyzed by Western blotting (lanes 14) or immunoprecip-itated with the anti-myc antibody prior to Western blotting (lanes 58). The cytoplasmic C-terminus domain of M protein interacted withNC protein. The protein larger than NC protein marked by the thinarrow is the immunoglobulin heavy chain (lower panel). More thanone band was detected when the plasmid encoding the cytoplasmic C-terminus domain of M protein was expressed, possibly due to samplepreparation without boiling treatment. (b) Vero E6 cells weretransfected with vector alone (lanes 1 and 5), plasmid encoding thefirst 100 amino acids of M protein with a V5 tag (lanes 2 and 6),plasmid encoding myc-NC (lanes 3 and 7), or co-transfected with bothplasmids (lanes 4 and 8). Cell lysates were directly analyzed byWestern blotting (lanes 14) or immunoprecipitated with the anti-mycantibody prior to Western blotting (lanes 58). M protein without itscytoplasmic domain did not interact with NC protein. The proteinlarger than NC protein marked by the thin arrow is the immunoglob-ulin heavy chain. (c) Vero E6 cells were transfected with vector alone,plasmid encoding M42137 plus a V5 tag (or the plasmid encodingM44668 plus a V5 tag), plasmid encoding myc-NC, or co-transfected with two plasmids (M42137 and myc-NC, M44668and myc-NC). Cell lysates were directly analyzed by Western blotting(lanes 16) or immunoprecipitated with the anti-myc antibody prior toWestern blotting (lanes 712). M protein without its first hydrophobicdomain, but not M protein without its second hydrophobic domaininteracted with NC protein. (d) Vero E6 cells were transfected withvector alone, plasmid encoding M1170 with a V5 tag (or the plasmidencoding M4101135 plus a V5 tag, or the plasmid encodingM4136170 plus a V5 tag), plasmid encoding myc-NC, or co-transfected with two plasmids (M1170 and myc-NC, M4101135and myc-NC, M4136170 and myc-NC). Cell lysates were directlyanalyzed by Western blotting (lanes 18) or immunoprecipitated withthe anti-myc antibody prior to Western blotting (lanes 918). All ofthe M cytoplasmic domain-deleted mutants interacted with NCproteinbJ Biomed Sci (2008) 15:707717711123room temperature), then incubated with FITC-conjugatedsecondary antibody (e.g., goat anti-mouse IgG antibody,diluted 209) at 37?C for 30 min. Samples were againwashed with PBS three times (510 min each time at roomtemperature).DAPI(40,6-diamidino-2-phenylindole)(Merck, Germany) was used to stain DNA to localize thecell nuclei. Samples were then observed with confocalmicroscopy. To quantify the average percentage of co-localization, the Image J (NIH website) program was used25.ResultsM protein co-localization in cultured cellsSARS-CoV structural proteins localized in different sub-cellular positions when they were expressed individually(Fig. 1). When SARS-CoV M protein was co-expressedwith other structural proteins (S, E, or NC), they werealmostentirelyco-localizedwiththeotherproteins(Fig. 2a); R = 0.93, 0.90, 0.90 for co-localization of M andS proteins, M and NC proteins, and M and E proteins,respectively. This result suggests that M protein binds toother structural proteins in the cultured cells. When S andE, S and NC, or E and NC were co-expressed in the cells,onlypartialco-localizationwasobserved(Fig. 2b);R = 0.54, 0.70, 0.85 for co-localization of E and S pro-teins, E and NC proteins, and NC and S proteins,respectively.Binding between M and other structural proteinsToverifywhetherSARS-CoVMproteinandotherstructuralproteins could bind to each other within cells, we performeda co-immunoprecipitation experiment. The V5-tagged, full-length M protein and the myc-tagged E protein were co-expressed in Vero E6 cells by transient transfection. Aftertransfection, cell lysates were immunoprecipitated with theanti-myc antibody, followed by Western blotting using theanti-V5 antibody. As shown in Fig. 3a, the V5-tagged Mproteinwasimmunoprecipitatedbytheanti-mycantibodyinthe presence (lane 8), but not in the absence (lane 6), of Eprotein. This result further confirmed that M and E proteinswere bound to each other in the cultured cells. Similar co-immunoprecipitation experiments were performed to studythe interactions between M and S proteins (Fig. 3b), and Mand NC proteins (Fig. 3c). These results confirmed that Mprotein also bound S and NC proteins in the cultured cells.Under the same experimental conditions (Supplemen-tary Fig. 1a), the interaction between E and M but not theinteraction between E and S was detected. Similarly, theinteraction between NC and M but not the interactionbetween NC and E was demonstrated (SupplementaryFig. 1b). Moreover, the interaction between NC and M butnot the interaction between NC and S was detected (Sup-plementary Fig. 1c).Deletion mapping and M protein interactionswith other structural proteinsTo identify which region(s) of SARS-CoV M protein wereinteracting with NC protein, a deletion mapping experi-ment was performed. As shown in Fig. 4a, the V5-taggedM protein without the first 100 amino acids was immu-noprecipitated by the anti-myc antibody in the presence(lane 8), but not in the absence (lane 6) of NC protein,while the first 100 amino acids of M protein with V5 tagcould not be immunoprecipitated by the anti-myc antibodyin the presence of the NC protein (Fig. 4b). This resultindicates that the cytoplasmic C-terminus domain ofSARS-CoV M protein was responsible for the binding withNC protein. To determine whether different topologies ofthe C-terminus domain of M protein could still interactwith NC protein, the first (amino acids 2137) or the sec-ond (amino acids 4668) transmembrane domain of Mprotein was deleted. M protein without the first trans-membrane domain still left its C-terminus domain in thecytoplasm and it was not glycosylated, while M proteinwithout the second transmembrane domain left its C-ter-minusdomainintheendoplasmicreticulumlumen(Supplementary Fig. 2). As shown in Fig. 4c, M proteinwithout the first transmembrane domain interacted with NCprotein while M protein without the second transmembranedomain did not. To identify the regions within the cyto-plasmic C-terminus domain of M protein responsible forbinding to NC protein, different regions (amino acids 101135, 136170, and 171221) within the cytoplasmic C-terminus domain of M protein were deleted separately. Asshown in Fig. 4d, all three M deletion mutants still inter-acted with NC protein. Co-localization between thecytoplasmic C-terminus domain of M protein and NCprotein was also demonstrated (upper panel, Fig. 7).Deletion mapping was also performed to identify whichdomains of M protein interacted with E protein. The C-terminal fragment from amino acids 171221 of M proteinwas truncated first. As shown in Fig. 5a, this truncated Mprotein still bound E protein. The first 50 amino acids ofthis truncated M protein were further deleted and stillinteracted with E protein. M protein without this region(amino acids 51170) still interacted with E protein. Theseresults suggest that multiple regions within M proteininteracted with E protein. The second and third trans-membrane regions (amino acids 4668 and 78100) of Mprotein by themselves, penetrated into the cell membrane,712J Biomed Sci (2008) 15:707717123while the first transmembrane region (amino acids 1436)was stabilized by interaction with the other transmembranesegments 25. To determine whether the second or thirdtransmembrane region alone interacted with E protein,these two transmembrane regions were fused with the first115 amino acids of HCV core protein separately 25. Asshown in Fig. 5b, the second or the third transmembraneregion of M protein alone was sufficient to bind E protein.Furthermore, the cytoplasmic C-terminus domain of Mprotein (i.e. M protein without its first 100 amino acids)Fig. 5 Immunoprecipitation and Western blotting analyses of SARS-CoV M protein fragment interactions with E protein. (a) Interactionsbetween SARS-CoV E and different M fragments. Vero E6 cells weretransfected with vector alone, with the plasmid encoding M1170with a V5 tag (or the plasmid encoding M51170 plus a V5 tag, or theplasmid encoding M451170 plus a V5 tag), plasmid encoding myc-E, or co-transfected with two plasmids (M1170 and myc-E, M51170 and myc-E, M451170 and myc-E). Cell lysates were directlyanalyzed by Western blotting (lanes 18) or immunoprecipitated withthe anti-myc antibody prior to Western blotting (lanes 918). (b) VeroE6 cells were transfected with the plasmid encoding myc-E protein,plasmid encoding the first 115 amino acids of HCV core protein andM protein amino acids 4668 plus a V5 tag (the plasmid encoding thefirst 115 amino acids of HCV core protein and M protein amino acids78100 plus a V5 tag), or co-transfected with two plasmids. Celllysates were directly analyzed by Western blotting (lanes 14) orimmunoprecipitated with the anti-V5 antibody prior to Westernblotting (lanes 58). Either the second or the third transmembranedomain of M protein was sufficient for interaction with E protein. (c)Interactions between SARS-CoV E and the cytoplasmic C-terminusdomain of M protein. Vero E6 cells were transfected with vectoralone, plasmid encoding myc-E protein, plasmid encoding M proteinwithout the first 100 amino acids plus a V5 tag, or co-transfected withboth plasmids. Cell lysates were directly analyzed by Westernblotting (lanes 14) or immunoprecipitated with the anti-V5 antibodyprior to Western blotting (lanes 58) (lower panel). More than oneband was detected when the plasmid encoding the cytoplasmic C-terminus domain of M protein was expressed, possibly due to samplepreparation without boiling treatmentJ Biomed Sci (2008) 15:707717713123also interacted with E protein (Fig. 5c). In this case, theunglycosylated, but not glycosylated, E protein was pref-erentially immunoprecipitated. Co-localization between thecytoplasmic C-terminus domain of M protein and E proteinwas also demonstrated (middle panel, Fig. 7).The M mutant constructs were also used to map thebinding region(s) of M protein with S protein. As shown inFig. 6a, M protein containing only amino acids 51170interacted with S protein. M protein without this regionalso interacted with S protein (Fig. 6b). Again, the cyto-plasmic C-terminus domain of M protein was alsosufficient to interact with S protein (Fig. 6c). These resultssuggest that multiple regions within M protein interactedwith S protein. Co-localization between the cytoplasmic C-terminus domain of M protein and S protein was alsodemonstrated (lower panel of Fig. 7).Fig. 6 M protein interaction with S protein. (a) Vero E6 cells weretransfected with vector alone (lanes 1 and 5), plasmid encoding M51170 with a V5 tag (lanes 2 and 6), plasmid encoding myc-S (lanes 3and 7), or co-transfected with both plasmids (lanes 4 and 8). Celllysates were directly analyzed by Western blotting (lanes 14) orimmunoprecipitated with the anti-myc antibody prior to Westernblotting (lanes 58). (b) Similar to (a), except the plasmid encodingM451170 plus a V5 tag was used to replace the plasmid encodingM51170 with a V5 tag. (c) Interactions between SARS-CoV S andcytoplasmic C-terminus domain of M protein. Similar to (a), exceptthe plasmid encoding M protein without the first 100 amino acids plusa V5 tag was used to replace the plasmid encoding M51170 with aV5 tag (lower panel). More than one band was detected when theplasmid encoding cytoplasmic domain of M protein was expressedpossibly due to the sample preparation without boiling treatment. Theresults show that multiple regions of M protein interacted with Sprotein714J Biomed Sci (2008) 15:707717123DiscussionSARS-CoV structural proteins (S, E, M, and NC) localizedto different subcellular positions when they were expressedindividually (Fig. 1), similar to the results of a previousreport 30. SARS-CoV M protein co-localized almostentirely with S, E, or NC proteins when they were co-expressed within the cells (Fig. 2a). On the other hand,only partial co-localization was observed when S and E, Sand NC, or E and NC were co-expressed in the cells(Fig. 2b). Furthermore, the interactions between M and theother structural proteins were demonstrated by co-immu-noprecipitation (Fig. 3). The interactions of E and S, E andNC, NC and S proteins were not demonstrated by co-immunoprecipitation (Supplementary Fig. 1). These resultssuggest that SARS-CoV M protein plays a pivotal role invirus assembly. Interactions of S and E, S and NC, or E andNC could occur after binding with M protein.Previous studies reported that virus-like particles (VLP)formed when either SARS-CoV M and E proteins 31 orM and NC proteins 32 were co-expressed in cells. Wehypothesize that M protein plays a crucial role in virusassembly and our study results support this hypothesis.SARS-CoV M protein appears to be a triple-spanningmembrane protein 33, while NC protein is a cytoplasmicprotein. Due to the topology of these two proteins, Mprotein is supposed to interact with NC through its cyto-plasmic C-terminus domain. Indeed, as shown in Fig. 4,only the C-terminus domain of M protein residing in thecytoplasm interacted with NC protein. The result that threeM deletion mutants (M protein without amino acids 101135, 136170, or 171221) interacted with NC protein(Fig. 4d) suggests that almost the entire C-terminus domainof M protein is responsible for the interaction with NCprotein. Our results agree with those of previous reportsusing in vitro GST pull-downed assays 34 or yeast two-hybrid and surface plasmon resonance techniques 35 tostudy the interactions between SARS-CoV M and NCproteins.Fig. 7 Co-localization of M41100 protein and NC (upper panel), E(middle panel), and S (lower panel) proteins. Similar to Fig. 2a,except the plasmid encoding M protein without the first 100 aminoacids plus a V5 tag was used to replace the plasmid encoding Mprotein with a V5 tagFig. 8 A proposed model for the interactions between the M proteinand other structural proteins of SARS-CoV. M protein probablyinteracts with unglycosylated E protein through cytoplasmic C-terminus regions first. After glycosylation of E protein, interactionbetween M and E proteins are possibly through transmembraneregions, and the freed cytoplasmic region of M protein will theninteract with NC protein. S protein could incorporate into virus-likeparticles formed by M and E proteins through interacting withmultiple regions of M proteinJ Biomed Sci (2008) 15:707717715123That at least two transmembrane domains and thecytoplasmic domain of M protein are sufficient for theinteracting with E protein (Fig. 5) suggest extensiveinteractions between these two proteins. Compared withother constructs, the cytoplasmic C-terminus domain of Mprotein preferentially immunoprecipitated unglycosylated,but not glycosylated, E protein (Fig. 5c). This result sup-ports a previously reported model for the topology of Eprotein 36: unglycosylated E protein leaves both its N-and C-termini in the cytoplasm while glycosylated E pro-tein leaves both its N- and C-termini in the endoplasmicreticulum lumen.Similar to the interactions of M and S proteins inanother coronaviruses 37, SARS-CoV M protein inter-acted with S protein through multiple regions (Fig. 6). Amodel for the interactions between SARS-CoV M proteinand other structural proteins was proposed (Fig. 8). WhileSARS-CoV structural proteins (S, E, M, and NC) reside indifferent subcellular locales, M protein brings other struc-tural proteins (NC, S, and E) together through interactingwith them. After that, interactions of S and E, S and NC, orE and NC occur.AcknowledgementThis work has been supported by grants fromTzu Chi University (TCIRP95002-01 and TCIRP96004-05) and fromNational Science Council of Taiwan (NSC 96-3112-B-320-001) toDr. Shih-Yen Lo.References1. Poon LL, Guan Y, Nicholls JM, Yuen KY, Peiris JS (2004) Theaetiology, origins, and diagnosis of severe acute respiratorysyndrome. Lancet Infect Dis 4:6636712. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R,Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen MH, TongS, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D,Erdman DD, Peret TC, Burns C, Ksiazek TG, Rollin PE, SanchezA, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Ras-mussen M, Fouchier R, Gunther S, Osterhaus AD, Drosten C,Pallansch MA, Anderson LJ, Bellini WJ (2003) Characterizationof a novel coronavirus associated with severe acute respiratorysyndrome. Science 300:139413993. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, Nicholls J,Yee WK, Yan WW, Cheung MT, Cheng VC, Chan KH, TsangDN, Yung RW, Ng TK, Yuen KY, SARS study group (2003)Coronavirus as a possible cause of severe acute respiratory syn-drome. Lancet 361:131913254. Kuiken T, Fouchier RA, Schutten M, Rimmelzwaan GF, vanAmerongen G, van Riel D, Laman JD, de Jong T, van DoornumG, Lim W, Ling AE, Chan PK, Tam JS, Zambon MC, Gopal R,Drosten C, van der Werf S, Escriou N, Manuguerra JC, Stohr K,Peiris JS, Osterhaus AD (2003) Newly discovered coronavirus asthe primary cause of severe acute respiratory syndrome. Lancet362:2632705. Gu J, Gong E, Zhang B, Zheng J, Gao Z, Zhong Y, Zou W, ZhanJ, Wang S, Xie Z, Zhuang H, Wu B, Zhong H, Shao H, Fang W,Gao D, Pei F, Li X, He Z, Xu D, Shi X, Anderson VM, Leong AS(2005) Multiple organ infection and the pathogenesis of SARS. JExp Med 202:4154246. Wang H, Rao S, Jiang C (2007) Molecular pathogenesis of severeacute respiratory syndrome. Microbes Infect 9:1191267. Enserink M (2003) Infectious diseases. Clues to the animal ori-gins of SARS. Science 300:13518. Holmes KV, Enjuanes L (2003) Virology. The SARS coronavi-rus: a postgenomic era. Science 300:137713789. Lai M (2003) SARS virus: the beginning of the unraveling of anew coronavirus. J Biomed Sci 10:66467510. Holmes K, Lai MM (1996) Coronaviridae: the viruses and theirreplication. In: Fields BN, Knipe DM, Howley PM (eds) Fieldsvirology, vol 1. Lippincott-Raven Publishers, Philadelphia, pp1075109311. Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S,Weissbrich B, Snijder EJ, Rabenau H, Doerr HW, GorbalenyaAE, Ziebuhr J (2003) Mechanisms and enzymes involved inSARS coronavirus genome expression. J Gen Virol 84:2305231512. Nguyen VP, Hogue BG (1997) Protein interactions during coro-navirus assembly. J Virol 71:9278928413. Nguyen VP, Hogue BG (1998) Coronavirus envelope glycopro-tein assembly complexes. Adv Exp Med Biol 440:36136514. Vennema H, Godeke GJ, Rossen JW, Voorhout WF, HorzinekMC, Opstelten DJ, Rottier PJ (1996) Nucleocapsid-independentassembly of coronavirus-like particles by co-expression of viralenvelope protein genes. EMBO J 15:2020202815. de Haan CA, Kuo L, Masters PS, Vennema H, Rottier PJ (1998)Coronavirus particle assembly: primary structure requirements ofthe membrane protein. J Virol 72:6838685016. Narayanan K, Maeda A, Maeda J, Makino S (2000) Character-ization of the coronavirus M protein and nucleocapsid interactionin infected cells. J Virol 74:8127813417. Godeke GJ, de Haan CA, Rossen JW, Vennema H, Rottier PJ(2000) Assembly of spikes into coronavirus particles is mediatedby the carboxy-terminal domain of the spike protein. J Virol74:1566157118. Opstelten DJ, Raamsman MJ, Wolfs K, Horzinek MC, Rottier PJ(1995)Envelopeglycoproteininteractionsincoronavirusassembly. J Cell Biol 131:33934919. Rottier P, Brandenburg D, Armstrong J, van der Zeijst B, WarrenG (1984) In vitro assembly of the murine coronavirus membraneprotein E1. Adv Exp Med Biol 173:536420. Rottier PJ, Welling GW, Welling-Wester S, Niesters HG, LenstraJA, Van der Zeijst BA (1986) Predicted membrane topology ofthe coronavirus protein E1. Biochemistry 25:1335133921. Stertz S, Reichelt M, Spiegel M, Kuri T, Martinez-Sobrido L,Garcia-Sastre A, Weber F, Kochs G (2007) The intracellular sitesof early replication and budding of SARS-coronavirus. Virology361:30431522. McBride CE, Li J, Machamer CE (2007) The cytoplasmic tail ofthe severe acute respiratory syndrome coronavirus spike proteincontains a novel endoplasmic reticulum retrieval signal that bindsCOPI and promotes interaction with membrane protein. J Virol81:2418242823. Hsieh PK, Chang SC, Huang CC, Lee TT, Hsiao CW, Kou YH,Chen IY, Chang CK, Huang TH, Chang MF (2005) Assembly ofsevere acute respiratory syndrome coronavirus RNA packagingsignal into virus-like particles is nucleocapsid dependent. J Virol79:138481385524. Lee YN, Chen LK, Ma HC, Yang HH, Li HP, Lo SY (2005)Thermal aggregation of SARS-CoV membrane protein. J VirolMethods 129:15216125. Ma HC, Fang CP, Hsieh YC, Chen SC, Li HC, Lo SY (2008)Expression and membrane integration of SARS-CoV M protein. JBiomed Sci 15:301310716J Biomed Sci (2008) 15:70771712326. Ma HC, Ku YY, Hsieh YC, Lo SY (2007) Characterization of thecleavage of signal peptide at the C-terminus of hepatitis C viruscore protein by signal peptide peptidase. J Biomed Sci 14:314127. Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotictransient-expression system based on recombinant vaccinia virusthat synthesizes bacteriophage T7 RNA polymerase. Proc NatlAcad Sci USA 83:8122812628. Ma HC, Ke CH, Hsieh TY, Lo SY (2002) The first hydrophobicdomain of the hepatitis C virus E1 protein is important forinteraction with the capsid protein. J Gen Virol 83:3085309229. Ma HC, Lin TW, Li H, Iguchi-Ariga SM, Ariga H, Chuang YL,Ou JH, Lo SY (2008) Hepatitis C virus ARFP/F protein interactswith cellular MM-1 protein and enhances the gene trans-activa-tion activity of c-Myc. J Biomed Sci 15:41742530. Nal B, Chan C, Kien F, Siu L, Tse J, Chu K, Kam J, Staropoli I,Crescenzo-Chaigne B, Escriou N, van der Werf S, Yuen KY,Altmeyer R (2005) Differential maturation and subcellularlocalization of severe acute respiratory syndrome coronavirussurface proteins S, M and E. J Gen Virol 86:1423143431. Ho Y, Lin PH, Liu CY, Lee SP, Chao YC (2004) Assembly ofhuman severe acute respiratory syndrome coronavirus-like par-ticles. Biochem Biophys Res Commun 318:83383832. Huang Y, Yang ZY, Kong WP, Nabel GJ (2004) Generation ofsyntheticsevereacuterespiratorysyndromecoronaviruspseudoparticles: implications for assembly and vaccine produc-tion. J Virol 78:125571256533. Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classifi-cation and secondary structure prediction system for membraneproteins. Bioinformatics 14:37837934. Fang X, Ye L, Timani KA, Li S, Zen Y, Zhao M, Zheng H, Wu Z(2005) Peptide domain involved in the interaction betweenmembrane protein and nucleocapsid protein of SARS-associatedcoronavirus. J Biochem Mol Biol 38:38138535. Luo H, Wu D, Shen C, Chen K, Shen X, Jiang H (2006) Severeacuterespiratorysyndromecoronavirusmembraneproteininteracts with nucleocapsid protein mostly through their carboxyltermini by electrostatic attraction. Int J Biochem Cell Biol38:58959936. Yuan Q, Liao Y, Torres J, Tam JP, Liu DX (2006) Biochemicalevidence for the presence of mixed membrane topologies of thesevere acute respiratory syndrome coronavirus envelope proteinexpressed in mammalian cells. FEBS Lett 580:3192320037. de Haan CA, Smeets M, Vernooij F, Vennema H, Rottier PJ(1999) Mapping of the coronavirus membrane protein domainsinvolved in interaction with the spike protein. J Virol 73:74417452J Biomed Sci (2008) 15:707717717123