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Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells
G S Marshall1, D P Fenger, G G Stout
1Department of Pediatrics, University of Louisville School of Medicine, Kentucky 40292, USA.
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.
Insights
Human cytomegalovirus (HCMV) glycoprotein B (gB) processing in A549 cells occurs late in the Golgi apparatus. This processing is crucial for understanding the immunogenicity of HCMV recombinant adenovirus vaccines.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Human cytomegalovirus (HCMV) glycoprotein B (gB) is essential for viral entry.
- Recombinant adenoviruses expressing HCMV gB are investigated as live-recombinant vaccines.
- Intracellular processing of gB can impact vaccine immunogenicity.
Purpose of the Study:
- To investigate the intracellular processing of HCMV gB expressed by a recombinant adenovirus (Ad-gB) in A549 cells.
- To determine the cellular location and characteristics of gB processing events.
- To assess the implications of gB processing for vaccine development.
Main Methods:
- Pulse-chase radiolabelling of Ad-gB infected A549 cells with [35S]methionine and [3H]mannose.
- Inhibition studies using brefeldin A to identify processing compartments.
- Endo-beta-N-acetylglucosaminidase H (Endo-H) sensitivity assays to analyze oligosaccharide processing.
- Comparison with gB processing in HCMV-infected human fibroblasts.
Main Results:
- gB cleavage into gp93 and gp55 subunits occurred, peaking after a 3-hour chase.
- Processing was inhibited by brefeldin A, indicating a late Golgi or post-Golgi event.
- Uncleaved gB remained Endo-H sensitive, suggesting retention in the endoplasmic reticulum with high-mannose oligosaccharides.
- Swainsonine treatment revealed differential oligosaccharide processing, with one site becoming Endo-H resistant.
- The gp93 subunit was detected using [3H]mannose labelling.
- gB cleavage was not observed in Ad-gB infected human fibroblasts, unlike native gB processing.
Conclusions:
- HCMV gB processing in Ad-gB infected A549 cells resembles native gB processing.
- Processing occurs as a late Golgi or post-Golgi event, involving modification of N-linked oligosaccharides.
- The observed processing differences in fibroblasts warrant further investigation for vaccine applications.