FIG. 9.

Rej does not facilitate the transport of unspliced cytoplasmic RNA in human 293 cells but is absolutely required for JSRV poly-Gag protein synthesis. 293 cells (10⁶) were transfected with 14 μg of pCMV2JS21, pCMV2JS21 SUΔ13-52, or pCDNA3.1; at 40 h posttransfection, the cells were harvested and total RNA or RNA from nuclei and cytoplasm was extracted. (A) Total, nuclear, and cytoplasmic RNAs from equivalent numbers of cells analyzed by agarose gel electrophoresis and Northern blot hybridization with a JSRV gag probe. Bands corresponding to the unspliced 7.5-kb and the 6.5-kb prematurely polyadenylated JSRV genome RNAs are shown (arrows, top panel). The blots were stripped and rehybridized with a GAPDH probe (1.2-kb band, arrow, bottom panel). (B) Western blot analysis of cell lysate fractions 1 to 6 of panel A with antibodies that detect β-tubulin (55 kDa, as a cytoplasmic marker) and lamin A/C (74 kDa, as a nuclear marker) was used to control for nuclear/cytoplasmic separation. Cell lysates were run on 10% SDS-polyacrylamide gels and visualized by immunoblotting; a 55-kDa specific band corresponding to β-tubulin was detected only in cytoplasmic fractions (lanes 1 and 3, top panel), whereas a 74-kDa specific band corresponding to lamin A/C was detected only in nuclear fractions (lanes 2 and 4, bottom panel); both proteins were detected in the total fractions (lanes 5 and 6, top and bottom panels). (C) Cell lysates at 40 h posttransfection from 293 cells transiently transfected with 14 μg of the plasmids indicated were analyzed by Western blotting with anti-JSRV CA monoclonal antibody. Gag polyprotein (74 kDa) was expressed from pCMV2JS21 (Gag, arrow, lane 2) but not from pCMV2JS21 SUΔ13-52 (lane 3) or pCDNA3.1 (lane 1). A nonspecific protein (NSP) that migrated faster than the JSRV poly-Gag protein was detected in all lanes and was used to show equivalent loading of the cell lysates (60 kDa, NSP, arrow).