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 studi ed in human A549 cells as processing events could affect immunogenicit y 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, s uggesting that processing normally occurs as a late Golgi or post-Golg i event, Uncleaved gp130 remained completely sensitive to endo-beta-N- acetylglucosaminidase H (Endo-H) in untreated cells following long cha se periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endo plasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated an d untreated cells was consistent with glycosylation at all three poten tial sites, with two oligosaccharides remaining sensitive to Endo-H an d 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]mann ose. No cleavage of gp130 was observed in analogous pulse-chase radiol abelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB mole cule, Processing of gB in recombinant adenovirus-infected A549 cells w as generally similar to that previously reported for native gB in HCMV -infected fibroblasts.