Figure 1

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 (M_N-FLAG) or a glycosylation-deficient M (M_N4QN-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 M_Insert and M_{N4Q 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 M_V186N and M_L205N were in vitro translated and analysed as described above.