Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus

B R Clark, J A Zaia, L Balce-Directo, Y P Ting
1984 Journal of Virology  
A guanidinium chloride extract of [3H]glucosamine-and [35S]methionine-labeled virions plus dense bodies of human cytomegalovirus (Towne) was separated by reverse-phase high-pressure liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eluate revealed the major peak to be a glycoprotein with a relative mass of 64,000. This glycoprotein (HCMVgp64) was characterized by amino acid analysis and a high-pressure liquid chromatographic map of its tryptic peptides. A
more » ... lypeptide with an Mr (relative mass) of between 64,000 and 69,000 is a major structural protein of human cytomegalovirus (HCMV) (4-9). Immune precipitation of this protein occurs upon reaction of virion components with acute-and convalescence-phase human sera obtained after natural HCMV infection (6) . A monoclonal antibody that is specific for this viral product neutralizes virus infectivity and binds to the plasma membrane of HCMV-infected cells (7) . Monolayer cultures of strain 350Q human foreskin fibroblasts (10) were grown in 495-cm2 roller bottles and infected on day 7 with HCMV at a multiplicity of infection of 0.1. Roller cultures were maintained in Dulbecco modified Eagle medium with 10% fetal calf serum (DMEM-10) at 37°C, and the extracellular fluid was removed at 96, 144, and 192 h after infection and replaced with fresh DMEM-10. Virions 1,500 -1,000 q) Q) 50. Q) (x50 and dense bodies were collected from extracellular fluid by gradient centrifugation through 20 to 70% sucrose followed by gradient centrifugation through 0 to 40% potassium tartrate-30 to 0% glycerol (1, 9). The band containing virions plus dense bodies (HCMV-db) was collected, washed three times in Tris-buffered saline, suspended in Tris-buffered saline, and frozen at -80°C. For radiolabeling of virions and dense bodies, roller bottles of cells infected with HCMV were pulsed with either [35S]methionine (New England Nuclear Corp., Boston, Mass.; 1,175 Ci/mmol) or [3H]glucosamine (Amersham Corp., Arlington Heights, Ill.; 29 Ci/mmol) at 5 ,uCi/ml of medium. Labeled HCMV-db was harvested from the extracellular fluid at 96, 144, and 192 h after infection as described above, and at 96 and 144 h the isotope-containing superna-
doi:10.1128/jvi.49.1.279-282.1984 fatcat:62qjmmgvvfd4rkz2qkrvr5bhsy