FIG. 3.

In vitro expression of middle and small surface proteins. Coupled eukaryotic in vitro transcription and translation reactions were carried out with expression plasmids driving the synthesis of wild-type (WT) and variant (F3 and F4) middle and small surface proteins with the T7 RNA polymerase promoter. All reactions were also carried out with canine microsomal membranes (+m) for posttranslational modifications. [³⁵S]methionine-radiolabelled proteins were analyzed by SDS-gel electrophoresis and autoradiography. The S proteins were expressed as unglycosylated and once-glycosylated peptides, with the exception of F4, for which once-glycosylated S protein could not be detected. Double-glycosylated S protein with a predicted size of about 30 kDa might be overlapping with the band seen for nonglycosylated M protein. The M proteins showed an additional double-glycosylated form due to an additional glycosylation site in the pre-S2 domain. Lane St, molecular mass markers. All autoradiograms were scanned with a Mustek Twain hand scanner and modified with program iPhoto Plus (version 1.1; U-Lead Systems, Inc.).