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-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O 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 gp 130 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 [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 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.
Marshall
G S
GS
Department of Pediatrics, University of Louisville School of Medicine, Kentucky 40292, USA.
Fenger
D P
DP
Stout
G G
GG
Knights
M E
ME
Hunt
L A
LA
D000260:Adenoviruses, Human / Q000235:genetics; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D003587:Cytomegalovirus / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017026:Swainsonine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D020126:Brefeldin A; D010450:Endopeptidases; D017026:Swainsonine