FIG. 5.

The alternatively spliced env transcripts encode Rej activity. (A) Schematic representation of the doubly spliced env cDNA expression vectors: ΔGPds-envcDNA2 and ΔGPds-envcDNA4 are shown relative to the other expression plasmids shown in Fig. 1. (B) 293T cells were transfected or cotransfected with the plasmids indicated, and at 40 h posttransfection, whole-cell lysates were analyzed on 7.5% SDS-polyacrylamide gels, followed by Western blot analysis with a monoclonal antibody to JSRV CA protein. The arrow indicates the cellular JSRV Gag polyprotein (74 kDa, top panel). Both doubly spliced expression constructs complemented pTN3 for Gag production equivalently to ΔGP and ΔGPSP. In pTN3-transfected cells, a slightly slower-migrating Gag-specific band was also detected, but it was distinct from the Gag polyprotein precursor. β-Tubulin (55 kDa) was used to control for equivalent loading of the cell lysates (bottom panel). (C) Northern blot hybridization analysis using a JSRV env probe indicated the authentic expression of env transcripts from ΔGP (2.4 kb, shown by an arrow) and the truncated env signal peptide from ΔGPSP or the ΔGP-spliced env transcripts expressed from ΔGPds-envcDNA2 and ΔGPds-envcDNA4 (0.7 kb, shown by an arrow).