Fig. 4

Comparison of WT and putative glyco-gag-mutated KoRVs in viral production. a DERSE cells were infected with WT and glyco-gag-mutated (gg-) KoRVs produced from 293T cells transfected with pKoRV522 and pKoRV gg-. Gag in the cell lysates and media were detected by western blots using anti-KoRV CA antibodies. Western blotting for beta-Tubulin in the cell lysates confirmed equal loading of samples (not shown). b Cell lysates from 293T cells transfected with pKoRV522 or from the M-MuLV infected cell line 43D were treated with PNGase (endoglycosidase) F to remove N-linked oligosaccharides, and Gag proteins were detected by SDS-PAGE and western blots using anti-KoRV CA and anti-MuLV p30 antibodies. The locations of the Pr65^gag Gag polyprotein precursor as well as a major cleavage product (Pr55^gag) are shown, as well as the corresponding proteins (Pr60^gag and Pr50^gag) for KoRV. M-MuLV glyco-gag (the primary translation product gPr80^gag as well as more slowly migrating forms with additional glycosylation) is indicated; endo F treatment reduced the size of gPr80^gag to 75 kDa [48]. Virus release efficiencies of gg- KoRV compared to WT KoRV (set at 1 in each experiment) are shown for infected DERSE (c) and 293T (d) cells. The release efficiency measurements resulted from at least three independent experiments; error bars indicate standard deviation.