Fig 3.

Western blot analysis. (A) MDBK cells were mock infected or infected with the WT BAC, gL STOP, and gL STOP Rev BoHV-4 strain (1 PFU/cell). Forty-eight hours later, cells were scraped and immunoblotted for virion components, using either a rabbit serum raised against whole virus (total BoHV-4) or MAbs specific for gp180, gB, and gH, as indicated. In the anti-gp180 blot, open and filled triangles indicate the specific 180-kDa protein and a background band, respectively. In the anti-gB blot, open and filled triangles indicate uncleaved and furin-cleaved gB, respectively. (B) Prediction of O-glycosylation sites for the complete BoHV-4 V.test strain gH protein sequence using the NetOglyc 3.1 algorithm. The shaded regions indicate the signal peptide and transmembrane region. The red line indicates the significance threshold. (C) Prediction of N-glycosylation sites for the complete BoHV-4 V.test strain gH protein sequence using the NetNglyc 1.0 algorithm. The shaded regions indicate the signal peptide and transmembrane region. The red line indicates the significance threshold. (D) MDBK cells were mock infected or infected with the WT BAC, gL STOP, and gL STOP Rev BoHV-4 strain (1 PFU/cell). Forty-eight hours later, cells were scraped and deglycosylated with EndoH or PNGase F and then immunoblotted for gH (MAb 9). (E) Purified virions were subjected to Western blotting with rabbit serum raised against whole virus (total BoHV-4) or MAbs specific for gp180, gB, and gH, as indicated. In the anti-gB blot, open and filled triangles indicate uncleaved and furin-cleaved gB, respectively. In the anti-gH blot (MAb 6), open and filled triangles indicate the specific 120-kDa gH protein and a background band, respectively. The position of an MM standard (kDa) is shown.