Fig 1.
Transient expression of a battery of sF glycoprotein constructs. A panel of NiV and HeV sF constructs and empty vector control (vector) were transfected into human 293T cells (A to D). At 48 h posttransfection, culture medium was harvested and cells were lysed and clarified by centrifugation. All constructs tested except wild type are codon optimized. (A) Expression of wild type and codon-optimized constructs without and with GCNt. (B) Expression of codon-optimized NiV and HeV sF and different mutants of NiV sF. (C) Expression of codon-optimized sF, sFGCNt, and different sF mutants of HeV. (D) Expression of codon-optimized NiV and HeV sFGCNt and different mutants of NiV sFGCNt. Bands migrating below F0 in panels C and D are most likely N-terminal degradation products (probing scheme using anti-S peptide). (E) Expression of GPI-anchored NiV F. The GPI-anchored NiV F construct was transfected into HeLa-USU (top) and HeLa-PLD (bottom) cells in a series of duplicate wells in 6-well tissue culture plates. At 48 h posttransfection, D-10 was replaced with either serum-free medium (D-0) or serum-free medium supplemented with 0.1 U of PLC (D-0+PLC). The culture supernatant containing PLC and the cells were harvested after 1 h; supernatants without PLC were harvested at 2, 16, and 22 h and cells were harvested at 22 h. A control D-10 supernatant was also harvested at 48 h. Cells were lysed and clarified by centrifugation. Cleared cell lysates and supernatants from panels A to E were precipitated with S-protein agarose. The precipitated proteins were resolved by 4 to 12% BT SDS-PAGE and detected by Western blotting with rabbit anti-S-peptide antibody. Sup, culture supernatant; Lys, cell lysate; dFp, fusion peptide deleted.