ABSTRACT
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.
Subject(s)
Cytomegalovirus/metabolism , Protein Precursors/metabolism , Protein Processing, Post-Translational , Viral Envelope Proteins/metabolism , Adenoviruses, Human/genetics , Brefeldin A , Cyclopentanes/pharmacology , Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Genetic Vectors/genetics , Glycosylation , Humans , Protein Synthesis Inhibitors/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Swainsonine/pharmacology , Tumor Cells, Cultured , Viral Envelope Proteins/geneticsABSTRACT
The envelope glycoprotein gB (gpUL55) is a candidate for inclusion in subunit cytomegalovirus (CMV) vaccines, although data on gB antibody responses after natural infection are limited. [35S]-labeled gB was partially purified from cells infected with an adenovirus recombinant expressing gB and used in radioimmunoprecipitation assays to characterize responses in solid organ transplant recipients with primary (n = 11) or secondary (n = 8) CMV infection. Seropositive transplant patients without evidence of infection (n = 5) and healthy seroconverters (n = 7) were also studied. gB antibody developed concurrently with CMV-specific IgG, IgM, and neutralizing activity in transplant patients with primary infection. Sustained boosts in gB antibody were seen in patients with secondary infection, and healthy seroconverters developed early gB responses. These data imply that gB antibody is an integral part of the humoral response to CMV infection, and, in view of experimental data regarding immunogenicity, support a role for gB in subunit vaccines.