Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus

Abstract

Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.

Constitutive expression of human cytomegalovirus glycoprotein B (gpUL55) with mutagenized carboxy-terminal hydrophobic domains

“…Monolayers were washed with cold PBS, harvested by scraping, the cells sedimented and extracts prepared by solubilization of the cell pellets in immunoprecipitation buffer (20 mM-Tris HC1 pH 9, 0.3 M-NaC1, 10 % glycerol, 1 mM-CaC12, 0.5mM-MgC12, 2mM-EDTA, 0'5% NP40, 0.5mM-PMSF, 100 units Trasylol/ml; 0.5ml/5× 106 cells). For immunoprecipitation Bogner et al, 1992) aliquots of cell extracts of comparable protein content or culture medium from labelled cells (freed of insoluble material by ultracentrifugation at 100000g for 2 h and 4 °C, mixed 1:1 with buffer) were precleared by incubation with Protein A-Sepharose CL4B beads (Sigma) prior to incubation overnight at 4 °C with gB-specific MAb (see above). Adsorption of immunocomplexes for 1.5 h at room temperature to Protein A-Sepharose CL4B beads coated with rabbit anti-mouse IgG (Dako) was followed by seven washing cycles of the beads with PBS plus 0" 1% NP40, 0.1% SDS and one washing cycle with distilled water.…”

“…Digestion with endoglycosidase H, peptide Nglycosidase F (PNGase F) or O-glycosidase was performed on immunoprecipitates according to the instructions of the manufacturer (Boehringer Mannheim; Bogner et al, 1992).…”

Stable constitutive expression of glycoprotein B (gpUL55) of human cytomegalovirus in permissive astrocytoma cells

“…Following seven cycles of washing the beads with PBS plus 0-1% NP40, 0.1% SDS and one washing cycle with distilled water the precipitates were subjected to SDS-PAGE (Laemmli, 1970) and consecutive fixation and fluorography (Bonner & Laskey, 1974) of the dried slab gels. For immunoblotting extracts prepared as described above were separated using SDS-PAGE under reducing or non-reducing conditions, transferred to nitrocellulose sheets and probed with gH-specific polyclonal antibody pAb gH (Bogner et al, 1992) and successively with HCMV pp65-specific MAb 28-19 (kindly provided by W. Britt) or with convalescent or hyperimmune serum, as described previously (Bogner et al, 1992).…”

“…Digestion with endoglycosidase H (endo H) and N-glucosidase F (PNGase F or pF) (Bogner et al, 1992;Radsak et al, 1990) was performed on immunoprecipitates of gB after one additional washing cycle with 100 mM-sodium phosphate pH 6, 0.05 % SDS, 0.5 % octylglycoside, 2 mM-EDTA, 0.5 mM-PMSF (glycosidase buffer) by incubation overnight at 37 °C in 80 gl of glycosidase buffer plus 0'1 M-2-mercaptoethanol. The instructions of the manufacturer (Boehringer) were followed for the enzyme concentrations.…”

“…Visualization of gB expression was carried out by indirect immunofluorescence with MAb 27-156 (Spaete et al, 1988) or glycoprotein H (gH)-specific MAb 14-4b (Bogner et al, 1992) and a secondary fluorescence-labelled rabbit anti-mouse IgG (Dakopatts) either on cells made permeable by acetone fixation for 15 rain at -20 °C or ceil surface fluorescence was studied on unfixed cells preincubated with 3% BSA in PBS or on cells fixed in 0.1% paraformaldehyde in PBS at room temperature (RT).…”