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

• Published in: Journal of general virology 1996-07, Vol.77 (7), p.1549-1557
• Main Authors: Marshall, Gary S, Fenger, Denice P, Stout, Gordon G, Knights, Mari E, Hunt, Lawrence A
• Format: Article
• Language: English
• Subjects: Adenoviruses, Human - genetics; Brefeldin A; Cyclopentanes - pharmacology; Cytomegalovirus - metabolism; Endopeptidases - metabolism; Enzyme Inhibitors - pharmacology; Genetic Vectors - genetics; Glycosylation; human cytomegalovirus; Humans; Protein Precursors - metabolism; Protein Processing, Post-Translational; Protein Synthesis Inhibitors - pharmacology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Swainsonine - pharmacology; Tumor Cells, Cultured; Viral Envelope Proteins - genetics; Viral Envelope Proteins - metabolism
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-77-7-1549

1 Department of Pediatrics and 2 Department of Microbiology & Immunology, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [ 35 S]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- - 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 [ 35 S]methionine-labelling but was easily detected along with gp55 after labelling with [ 3 H]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. Received 8 November 1995; accepted 13 March 1996.