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. 2001 Dec;75(24):12228-40.
doi: 10.1128/JVI.75.24.12228-12240.2001.

Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core

D Escors  1 , E Camafeita, J Ortego, H Laude, L Enjuanes
Affiliations

Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core

D Escors et al. J Virol. 2001 Dec.

Abstract

The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of (35)S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.

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Figures

FIG. 1
FIG. 1
TGEV M protein topologies in the virus envelope. Scheme of the topologies adopted by the M protein, the Nexo-Cendo topology (left) and the Nexo-Cexo topology (right), showing the MAbs used in this report and the approximate locations of the M protein epitopes recognized by them. The domain of the M protein that interacts with the nucleocapsid is indicated as a wide bar between residues 237 and 252. Ext, external surface of the virion; Int, virion interior. Arrows indicate approximate amino acid positions in the M protein.
FIG. 2
FIG. 2
Electron microscopy of negatively stained, purified TGEV virions and cores and estimations of the M-to-N molar ratios. (A) Electron microscopy pictures of sucrose-purified TGEV virions (left panel) and cores (right panel). Bars, 100 nm. (B) Left panel, SDS-PAGE analysis and silver staining of purified TGEV virions and cores. Arrows indicate the positions of the major structural proteins. Right panel, estimations of the M-to-N molar ratios in virions and cores presented as means ± SDs. The means of the M-to-N molar ratios were deduced from eight independent experiments.
FIG. 3
FIG. 3
Immunopurification of 35S-labeled TGEV virions. (A) SDS-PAGE and fluorography of immunopurified 35S-labeled TGEV virions using the indicated MAbs. Arrows indicate the positions of the TGEV structural proteins S, N, and M. −, absence of MAb.; IC, extract from infected cells. (B) Inhibition of immunopurification of labeled virions with the indicated concentrations of unlabeled purified TGEV virions, using MAbs 25.22, 3D.E3, and 9D.B4 as indicated. Control experiments were performed using concentrated MHV virions as an unspecific competitor at the highest concentration assayed. + or −, presence or absence of unlabeled MHV, respectively. Arrows indicate the positions of the structural proteins.
FIG. 4
FIG. 4
Expression of Mwt protein and deletion mutant M proteins. (A) Scheme of mutant M proteins cloned in the expression vector pcDNA3. The gray boxes indicate deletions. The numbers above the bars represent the amino acid positions immediately before and after the deletions. The approximate locations of the MAb binding sites in the M protein are indicated above the Mwt protein bar. (B) Western blot analysis of BHK-pAPN cell lysates expressing the indicated mutant M proteins using a polyvalent TGEV-specific antiserum. The M genes were cloned in plasmid pcDNA3 under the T7 polymerase promoter, and the vaccinia virus-expressing T7 polymerase was used to drive the expression. (C) Immunofluorescence microscopy patterns of BHK-pAPN cells transfected with pcDNA3 plasmids encoding Mwt and the indicated mutant M genes (left) using the M protein-specific MAbs indicated at the top. TGEV-infected ST cells were used as a positive expression control (top row).
FIG. 5
FIG. 5
Immunounification of 35S-labeled virions by MAbs. SDS-PAGE and fluorography of labeled TGEV virions immunopurified with MAb 25.22 (A), MAb 9D.B4 (B), and MAb 3D.E3 (C) were performed in the presence of increasing amounts of cell lysates from BHK-pAPN cells expressing Mwt (top row), MΔ1–50 (second row), MΔ253–262 (third row), and MΔ146–262 (bottom row). vT7, lysate of vT7-infected BHK-pAPN cells.
FIG. 6
FIG. 6
Mass spectrometry (MALDI-TOF) analysis of tryptic peptides released from the TGEV virion surface. MALDI-TOF spectra of trypsin (A), untreated TGEV virions (B), and TGEV virions treated with trypsin for 15 (C) or 45 (D) min at room temperature are shown. A domain of the spectrum that includes the predicted M peptide with an m/Z of 1,656.8 is shown in the figure.
FIG. 7
FIG. 7
Binding of M-specific MAbs to the virus surface. (A) Western blot analysis of the heavy and light antibody chains from MAbs specifically bound to the virion surface. IgG, purified immunoglobulin G. + NP-40 or − NP-40, presence or absence of NP-40 in virus preparations. (B) Western blot analysis of the heavy and light chains of the M-specific MAbs bound to the surfaces of purified virions (V) and their cores (C). Heavy (H) and light (L) immunoglobulin chains are indicated by arrows.
FIG. 8
FIG. 8
Susceptibility of TGEV M protein to deglycosylation. (A) Western blot of N-glycosidase F (Glyc. F)-treated virions in the presence (+) or absence (−) of the detergents NP-40 and SDS. (B) Deglycosylation kinetics of TGEV virion M protein by N-glycosidase F at the indicated times. (C) Western blot of endoglycosidase H (Endo H)-treated virions in the presence (+) or absence (−) of the detergents NP-40 and SDS. (D) Deglycosylation kinetics of TGEV virion M protein by glycosidase H at the indicated times.
FIG. 9
FIG. 9
Working model for the TGEV virion structure and structural dissociation. A working scheme of the chemical dissociation of TGEV virions compatible with all the experimental observations obtained is shown. According to this model, cores from purified virions were purified by removal of the lipid bilayer with the M protein in a Nexo-Cexo topology. Cores treated with high salt concentrations were disrupted, lost their M proteins, and became unstable, which led to release of the helical nucleocapsids that form the cores. M and M′, membrane protein molecules adopting Nexo-Cendo and Nexo-Cexo topologies, respectively.
FIG. 10
FIG. 10
M protein compositions of cores purified from glycosidase-treated virions. Shown are Western blots of virions and cores purified from virions treated with N-glycosidase glycosidase F (Glyco. F) (A) or endoglycosidase H (Endo H) (B).
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