Nuclear compartmentation of glycoprotein B of human cytomegalovirus

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

“…For propagation of strain AD169 of HCMV confluent monolayers of HFF cells (1"5 × 107) were infected with an m.o.i, of approximately 0"01 and serum concentration was lowered to 2 % ; for experimental infection an m.o.i, of about 3 for HFF and 5 for U373 cells was used. To determine infectious units, coverslip cultures were infected with the virus suspensions prior to examination at 48 h post-infection for early antigen (EA) production by indirect immunofluorescence using a commercial monoclonal antibody (MAb) (DuPont; Radsak et al, 1990). For quantification EA-positive nuclei were counted; the amount of virus inducing EA production in a single cell was defined as 1 infectious unit (IU).…”

“…Selection was initiated with geneticin at 800 lag/ml 2 days after transfection. Resistant clones emerging after 2 to 3 weeks were aspirated under the microscope with the tip of a micropipette and propagated prior to examination of gB expression by indirect immunofluorescence with the gB-specific MAb 27-156 (Spaete et al, 1990;Radsak et aL, 1990; generously provided by W. Britt, Birmingham, Ala., U.S.A.). Because selection by geneticin alone did not yield populations in which 100% of the cells expressed the gB polypeptide, subcloning by aspiration of single cells as described above was performed.…”

“…For radiolabelling prior to immunoprecipitation, the complete culture medium was replaced by labelling medium consisting of MEM lacking methionine plus [35S]methionine (50 to 100 laCi/ml; specific activity > 1000Ci/mmol; Amersham Buchler) (see Results). Monolayers (1-5 x 106 to 5 x 106 cells) were harvested by scraping, centrifuged and extracts prepared by dissolving the cell pellets in 20 mM-Tris-HC1 pH 9, 0.3 M-NaC1, 10% glycerol, 1 mM-CaC12, 0.5 mM-MgC12, 2 mM-EDTA, 0"5 % NP40, 0"5 mM-PMSF, 100 units Trasylol/ml (Radsak et al, 1990; 0"5 ml/5 x 106 cells). For immunoprecipitation (Radsak et al, 1990), samples of cell extracts of comparable protein content were precleared by incubation with Protein A-Sepharose CL4B beads (Pierce) prior to incubation overnight at RT with MAb 27-156 or 14-4b.…”

“…Monolayers (1-5 x 106 to 5 x 106 cells) were harvested by scraping, centrifuged and extracts prepared by dissolving the cell pellets in 20 mM-Tris-HC1 pH 9, 0.3 M-NaC1, 10% glycerol, 1 mM-CaC12, 0.5 mM-MgC12, 2 mM-EDTA, 0"5 % NP40, 0"5 mM-PMSF, 100 units Trasylol/ml (Radsak et al, 1990; 0"5 ml/5 x 106 cells). For immunoprecipitation (Radsak et al, 1990), samples of cell extracts of comparable protein content were precleared by incubation with Protein A-Sepharose CL4B beads (Pierce) prior to incubation overnight at RT with MAb 27-156 or 14-4b. Immunocomplexes were adsorbed for 1.5 h RT onto Protein A Sepharose CL4B beads coated with rabbit anti-mouse IgG (Dakopatts).…”

Human cytomegalovirus late-phase maturation is blocked by stably expressed UL32 antisense mRNA in astrocytoma cells.

“…As for the membrane component, the viral glycoprotein gB (gpUL55) can be detected in the inner nuclear membrane (Radsak et al, 1990) as well as in the final envelope and is thus a potential participant in these interactions.…”