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Abstract
The glycosylated membrane protein M of the severe acute respiratory syndrome associated coronavirus (SARS-CoV) is the main structural component of the virion and mediates assembly and budding of viral particles. The membrane topology of SARS-CoV M and the functional significance of its N-glycosylation are not completely understood as is its interaction with the surface glycoprotein S. Using biochemical and immunofluorescence analyses we found that M consists of a short glycosylated N-terminal ectodomain, three transmembrane segments and a long, immunogenic C-terminal endodomain. Although the N-glycosylation site of M seems to be highly conserved between group 1 and 3 coronaviruses, studies using a recombinant SARS-CoV expressing a glycosylation-deficient M revealed that N-glycosylation of M neither influence the shape of the virions nor their infectivity in cell culture. Further functional analysis of truncated M proteins showed that the N-terminal 134 amino acids comprising the three transmembrane domains are sufficient to mediate accumulation of M in the Golgi complex and to enforce recruitment of the viral spike protein S to the sites of virus assembly and budding in the ERGIC.
Figures
Analysis of the membrane topology of SARS-CoV M. A, in silico predictions of hydrophobic domains and potential transmembrane segments of M using various computer algorithms. Numbers in superscript refer to the position of the first and the last amino acids of potential transmembrane domains (white rectangles). B, Subconfluent Huh7 cells were transfected with plasmids encoding M (MN-FLAG) or a glycosylation-deficient M (MN4QN-FLAG) both N-terminally fused with a FLAG-peptide. Surface-staining (red fluorescence) and subsequent intracellular staining (green fluorescence) of M was performed 24 h posttransfection (p.t.) using a polyclonal α-FLAG and a fluorescence-labelled secondary antibody. C, M and the glycosylation mutants MInsert and MN4Q Insert were in vitro translated in the presence of canine microsomal membranes and metabolically labelled with [35S] PROMIX (Promega). Resultant proteins were digested with Endo H. Membrane-bound proteins were pelleted and subjected to SDS-PAGE analysis. Radioactive signals were visualized using Bioimager analyser (BAS-1000; Fuji). M0 – non-glycosylated M; M1 – mono-glycosylated M; M2 – di-glycosylated M. D, M and the glycosylation mutants MV186N and ML205N were in vitro translated and analysed as described above.
N-glycosylation of coronviral M. A, N-terminal amino acid sequences of M of selected coronaviruses were aligned and depicted using MacVector program. B, M and the glycosylation-deficient mutant MN3Q of HCoV-NL63 were in vitro translated and analyzed as described above. (Plasmid expressing HCoV-NL63 M protein was kindly provided by M. Müller, Bonn). C, Subconfluent Huh7 cells were transfected with plasmid encoding the glycosylation-deficient M (MN4QN-FLAG) using FuGENE according to the manufacturer's protocol. At 24 h p.t., cells were fixed, permeabilized and incubated with a polyclonal α-FLAG antibody and a rhodamine-coupled secondary antibody. ERGIC was stained using a monoclonal α-ERGIC primary antibody and a FITC-coupled secondary antibody. The merged image is shown on the right.
Characterization of recombinant SARS-CoV expressing a glycosylation-deficient M. A, Vero cells were infected with wild type recombinant SARS-CoV (wt rec SARS-CoV) or the mutant MN4Q recombinant SARS-CoV (MN4Q rec SARS-CoV) respectively with an moi of 3 pfu per cell. Cell lysates were harvested and subjected to Western blot analysis. Virus inactivation was achieved by treatment with 1% SDS and boiling of the samples. Proteins were separated by SDS-PAGE and blotted onto PVDF membranes. To detect viral structural proteins the membrane was incubated with a serum of an immunized rabbit or with the human monoclonal antibody S30. B, For electron microscopic analysis, Vero cells were infected with a MOI of 1 with wt rec SARS-CoV or MN4Q rec SARS-CoV, respectively. At 24 h p.i., viral particles were fixed, negatively stained and subjected to electron microscopic analysis.
Mapping the epitope of the human monoclonal α-M antibody S30. A, An exemplary schematic representation of M mutants used in this study. A FLAG-epitope was fused to the C-terminus or in addition to the N-terminus. B, M1–134 was in vitro translated in the presence of [35S] PROMIX and subsequently immunoprecipitated using human monoclonal α-M antibody S30. The precipitates were subjected to SDS-PAGE and radioactive signals were visualized by autoradiography. C and D, BHK-T7 cells were transfected with plasmids encoding various C-terminally truncated M mutants. At 24 h p.t., cells were lysed, sonicated and directly subjected to SDS-PAGE analysis followed by immunoblotting using polyclonal α-FLAG and human monoclonal α-M antibody S30 simultaneously. The proteins were visualized using an AF680-coupled α-rabbit and an IRDye800-coupled α-human antibody and the Odyssey™ Infrared Imaging System. Mc-g, complex glycosylated form of M. Arrowheads indicate the specific protein bands detected by α-M S30 antibody.
Intracellular distribution of M and M1–134. Huh7 cells were transfected with plasmids encoding M or M1–134 as described in Fig. 2. At 24 h p.t., cells were fixed, permeabilized and incubated with a polyclonal α-FLAG and a monoclonal α-Giantin antibody followed by a FITC-coupled α-rabbit and a rhodamine-coupled α-mouse antibody. The merged images are shown on the right hand side, respective proteins are given on the left hand side.
Coexpression of M and S. A, Huh7 cells were transfected with plasmid as described in Fig. 2. At 24 h p.t., cells were fixed, permeabilized and viral proteins were stained with a rabbit polyclonal α-S serum and a rhodamine-coupled α-rabbit secondary antibody. SARS-CoV (strain Frankfurt) infected Vero cells were fixed, permeabilized and incubated with the same antibodies as described above. B, Huh7 cells coexpressing MC or the truncated form M1–134 together with S were fixed, permeabilized and incubated with a rabbit α-S serum and a mouse monoclonal α-FLAG antibody followed by a FITC-coupled α-mouse and a rhodamine-coupled α-rabbit secondary antibody. The merged images are shown on the right hand side and respective, coexpressed proteins are given on the left hand side of the panels.
Membrane topology model of SARS-CoV M protein.
References
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- WHO Update: Outbreak of severe acute respiratory syndrome – worldwide, 2003. MMWR Morb Mortal Wkly Rep. 2003;52:241–246. - PubMed
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