Fig 3. Modifications of FFV bel2 with OVA do not significantly influence viral protein levels or infectivity.
A) Schematic representation of epitope replacements in Bet. Original protein sequences are shown in black; modifications in red. Epitope sequences are underlined. B) Cell lysates and C) enriched culture supernatants of 293T cells transfected with modified and wild-type proviruses were analyzed by immunoblotting. Cells and supernatants were harvested 2 d post-transfection and probed using polyclonal sera against the Gag matrix and Env transmembrane domains. Detection of Env and Gag in the particulate fraction represents specifically released virus particles. The Gag precursor (p52), cleaved mature Gag (p48), Env precursor (gp130Env), mature TM (gp48TM) and a cell lysate-associated transmembrane isoform (TMCL) are indicated by arrows. Proper protein loading of cell lysates was determined by probing for β-actin. D) CrFK and E) KE-R cells were infected with virus-containing supernatants harvested from 293T cells transfected with modified and wild-type proviruses. Supernatants were passaged in uninfected cells every two days. Viral titers were determined by titration on FeFAB cells. Cells were mock-infected with supernatant from pcDNA-transfected cells as a negative control. Titers are presented as mean values of three independent experiments. Error bars represent standard deviation of mean values.