FIG. 4.

Detection of BUNV glycoproteins on the cell surface. (A and B) Cell surface staining of nonpermeabilized cells. Cells were fixed with 4% paraformaldehyde and stained with anti-BUNV polyclonal antibody or anti-Gc MAb 742. Surface expression of viral glycoproteins was observed using a Delta Vision restoration microscope, and nuclei were stained blue with DAPI. (A) BSR-T7/5 cells were infected with wt BUNV or transfected with wt BUNV M segment cDNA (wt BUNM), BUNMΔGnCT (ΔGnCT), and BUNMΔGcCT (ΔGcCT) constructs, as indicated. Infected cells were stained with MAb 742, and the transfected cells were stained with anti-BUNV antibody. (B) BSR-T7/5 cells were transfected with BUNV M segment cDNA clones containing alanine substitutions in the Gn CT, as indicated. The cells were stained with anti-BUNV antibody. (C). Cell surface biotinylation of either wt BUNV-infected or alanine substitution mutant-transfected BSR-T7/5 cells. The biotinylated viral glycoproteins expressed on the cell surface were precipitated with anti-BUNV serum and separated in SDS-PAGE gels, and signals were revealed by chemiluminescence. No Gn protein was detected.