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. 2000 Feb;74(3):1566-71.
doi: 10.1128/jvi.74.3.1566-1571.2000.

Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein

G J Godeke  1 , C A de Haan, J W Rossen, H Vennema, P J Rottier
Affiliations

Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein

G J Godeke et al. J Virol. 2000 Feb.

Abstract

The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and endodomain. To this aim, we used the virus-like particle (VLP) system that we developed earlier for the mouse hepatitis virus strain A59 (MHV-A59) and which we describe now also for the unrelated coronavirus feline infectious peritonitis virus (FIPV; strain 79-1146). Two chimeric MHV-FIPV S proteins were constructed, consisting of the ectodomain of the one virus and the transmembrane and endodomain of the other. These proteins were tested for their incorporation into VLPs of either species. They were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated. Thus, the 64-terminal-residue sequence suffices to draw the 1308 (MHV)- or 1433 (FIPV)-amino-acid-long mature S protein into VLPs. Both chimeric S proteins appeared to cause cell fusion when expressed individually, suggesting that they were biologically fully active. This was indeed confirmed by incorporating one of the proteins into virions which thereby acquired a new host cell tropism, as will be reported elsewhere.

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Figures

FIG. 1
FIG. 1
(A) Spike constructs. MHV-A59 S was expressed from the plasmid pTUMS (32), and the FIPV strain 79-1146 S protein was expressed from pFIPVE2, which was made as follows. A 3′-terminal S fragment was prepared by ligating the XbaI-SalI fragment from pB1 (4) into pUC18, cutting with AccI and SalI, and religating after filling in with Klenow polymerase. From the resulting plasmid p3d, the XbaI-SalI fragment was isolated and used. A middle piece was prepared by isolating the PstI-XbaI fragment from pB1. This fragment and the 3′ XbaI-SalI fragment were ligated into p1A (4), which had been digested with PstI and SalI to give pFIPVE2. Chimeric protein FMS was expressed from pTFMS, which was constructed as follows. Plasmid p3d was digested with HindIII, filled in with Klenow enzyme, and ligated with BglII linkers, resulting in p3dHrB. After the plasmid was cut with StyI and BglII, an MHV S gene fragment was ligated into it; the fragment was prepared by digesting the S gene, obtained as a BamHI fragment from pDGE2 (31), with StyI and taking the small fragment. The resulting p3FM vector was cut with PstI and SalI; into it were ligated the XbaI-SalI fragment from p3d and the PstI-XbaI fragment from pB1. The chimeric gene was finally recloned as a BamHI fragment into pTUG3, resulting in pTFMS. Chimeric protein MFS was expressed from pTMFS, which was prepared starting with p3dHrB. This plasmid was cut with StyI and BamHI, and a BamHI-StyI fragment obtained from the MHV S BamHI gene described above was ligated into it. The chimeric S gene was recloned as a BamHI-SalI fragment into pTUG3 cut with the same enzymes. TM, transmembrane domain; ecto, ectodomain; endo, endodomain. (B) Carboxy-terminal sequences of the MHV-A59 and FIPV spike proteins. The 67 terminal residues of each protein are compared. The arrow indicates the junction point in the chimeric S constructs.
FIG. 2
FIG. 2
Expression of chimeric spike proteins. Parallel cultures of OST7-1 cells in 35-mm-diameter dishes were infected with vTF7-3 and transfected with plasmids encoding the wild-type and chimeric S proteins described in the legend to Fig. 1. Cells were incubated at 32°C. Starting at 4.5 h p.i., they were starved for 30 min in cysteine- and methionine-free minimal essential medium containing 10 mM HEPES (pH 7.2) without fetal bovine serum. The medium was then replaced by 600 μl of the same containing 100 μCi of 35S in vitro cell labeling mix (Amersham). After a 1-h labeling period, cells were washed with phosphate-buffered saline and solubilized in 1 ml of lysis buffer, TES (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA) containing 1% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride. Nuclei were removed from the cell lysates by centrifugation at 12,000 × g for 10 min at 4°C. For immunoprecipitations, 50-μl aliquots of lysate were diluted with 1 ml of detergent solution (50 mM Tris-HCl [pH 8.0], 62.5 mM EDTA, 0.5% Nonidet P-40, 0.5% Na deoxycholate), and 30 μl of 10% sodium dodecyl sulfate was added. MAbs were then added: 3 μl of hybridoma culture supernatant WA3.10 or 23F4.5, which recognizes the S protein of MHV (αSm) or FIPV (αSf), respectively. Following an overnight incubation at 4°C, immune complexes were adsorbed for 1 h to formalin-fixed Staphylococcus aureus cells (BRL Life Technologies) added as 45 μl of a 10% (wt/vol) suspension. Immune complexes were collected by centrifugation at 12,000 × g and washed three times with radioimmunoprecipitation assay buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 1% Na deoxycholate). Pellets were resuspended in 30 μl of Laemmli sample buffer, heated for 2.5 min at 95°C, and analyzed by electrophoresis in a sodium dodecyl sulfate–12.5% polyacrylamide gel followed by fluorography. MW, molecular mass.
FIG. 3
FIG. 3
Fusion properties of the chimeric spike proteins. Subconfluent monolayers of BHK-21 cells grown in 35-mm-diameter dishes were infected with vTF7-3 and transfected with the plasmids encoding MHV-A59 S (mS), FIPV S (fS), and the chimeric S proteins FMS and MFS. At 8 h p.i., the cells were overlaid with either LR7 cells (mouse L cells) or feline FCWF cells. Fusion was followed by light microscopy, and at 24 h p.i., pictures were taken.
FIG. 4
FIG. 4
Incorporation of chimeric FMS into MHV-based VLPs. Parallel cultures of OST7-1 cells in 35-mm-diameter dishes were infected with vTF7-3 and transfected with different combinations of plasmids as indicated (mS, mE, and mM represent plasmids encoding the wild-type MHV-A59 S, E, and M proteins, respectively; FMS and MFS refer to plasmids encoding the chimeric S proteins described in the legend to Fig. 1). Cells were incubated at 32°C and labeled from 5 to 8 h p.i. with 35S-amino acids (100 μCi/dish). Culture media (0.8 ml) were harvested, cleared by low-speed centrifugation, mixed with 2.3 ml of 67% sucrose in TM (10 mM Tris-HCl [pH 7.0], 10 mM MgCl2), and transferred into Beckman SW50.1 ultracentrifuge tubes. Each solution was overlaid with 1 ml of 48% sucrose, 0.5 ml of 40% sucrose, and 0.5 ml of 30% sucrose in TM, and the gradients were centrifuged at 36,000 rpm for 43 h. After centrifugation, a fraction consisting of the top 1 ml of each tube was collected. Virus particles were affinity purified from 150 μl of this fraction by addition of 25 μl of MAb J1.3 against the MHV M protein (αMm); 10 μl of MAb WA3.10, which is directed against an epitope in the MHV S ectodomain (αSm); or 3 μl of MAb 23F4.5, which recognizes an epitope in the FIPV S ectodomain (αSf). Samples were processed and analyzed as described for Fig. 2 except that the Staphylococcus aureus immune complexes were washed once with TM instead of three times with radioimmunoprecipitation assay buffer. At the left of the figure, mS and mS/gp90 indicate the positions of the uncleaved and cleaved forms of the S protein, respectively; mM and FMS mark the positions of the M protein and the chimeric S protein, respectively. Ab, antibody.
FIG. 5
FIG. 5
Incorporation of chimeric MFS into FIPV-based VLPs. Different plasmid combinations were expressed, the proteins were labeled, and the culture media were processed, all as described for Fig. 4. fS, fE, and fM refer to plasmids encoding the wild-type FIPV S, E, and M proteins, respectively; FMS and MFS refer to the chimeric constructs described in the legend to Fig. 1. The αFIPV serum (G73) was from an FIPV-infected cat. Ab, antibody.
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