Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-SE) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines, Cleavage of [S-35]methionine-labelled gp130 into gp93 and gp55 reached a maximum after a 3 h chase, Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event, Uncleaved gp130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance, The heavily glycosylated N-terminal gp93 subunit was not detected by [S-35]methionine-labelling but was easily detected along with gp55 after labelling with [H-3]mannose. No cleavage of gp130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule, Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.