FIG. 5.

Production of pseudoparticles by coexpression of heterologous and/or chimeric M and E genes of TGEV and BCV viruses. (a) Alignments of the sequences of mature TGEV and BCV M proteins (T and B, respectively) and of TGEV and BCV E genes (t and b, respectively). The predicted transmembrane segments are underlined in the TGEV sequences. Identical amino acids are in uppercase letters. In boldface are shown two short homologous regions selected to fuse TGEV and BCV sequences in the chimeric M or E gene constructs used in these experiments. Sequence data are from references 33 (TGEV M), 30 (BCV M), 19 (TGEV E), and 1 (BCV E). (b and c) Various pairs of homologous and chimeric constructs were expressed through the vT7-3 expression system. Particle assembly and secretion were assessed by monitoring the release of soluble, radiolabeled M material in the culture medium of transfected cells. Sedimentable material was collected by centrifugation through a glycerol cushion and analyzed by SDS–12% PAGE and autoradiography. The E chimera designated bt corresponds to the BCV amino-half sequence fused with the TGEV carboxy-half sequence, and tb is the reciprocal construct. The M chimera designated TBB corresponds to the TGEV N-terminal ectodomain fused with the remaining part of BCV M sequence, and vice versa for the BTT chimera. TBB and BTT chimeric proteins were expressed at equivalent levels as judged by immunoprecipitation assays of lysates of transfected cells with MAbs 25.22 and 3.60, respectively. Numbers on the left of panel b are molecular weights in thousands.