FIG. 8.
Membrane integration of deletion mutant forms of MHV and SARS-CoV nsp4. (A, D) Schematic representations of the MHV (A) and SARS-CoV (D) nsp4 C-terminal deletion mutant forms, with the hydrophobic domains presented as black rectangles and the EGFPglyc tag shown in gray. (B, E) vTF7-3-infected OST7-1 cells were transfected with the indicated constructs and expressed in the presence (+) or absence (−) of tunicamycin (TM). The cells were labeled with [35S]methionine from 5 to 6 h p.i., lysed, and processed for immunoprecipitation with anti-EGFP antiserum, followed by SDS-PAGE. The positions and masses (in kilodaltons) of the protein size markers are indicated at the left. Only the relevant portions of the gels are shown. The asterisks indicate the position of the glycosylated protein species. Below the panels, the observed presence or absence of N glycosylation is indicated by a plus or a minus sign, respectively (C, F). Models of the membrane structure of nsp4 of MHV (C) and SARS-CoV (F), with the hydrophobic domains presented as black rectangles.