No abstract

Cell surface expression of the human cytomegalovirus (HCMV) major envelope glycoprotein complex, gp55-116 (gB), was studied by using monoclonal antibodies and an HCMV gp55-116 (gB) recombinant vaccinia virus. HCMV-infected human fibroblasts and recombinant vaccinia virus-infected HeLa cells expressed three electrophoretically distinct proteins of Mr 170,000, 116,000, and 55,000 on their surface. These species have been previously identified within infected cells and purified virions. Two unique neutralizing epitopes were shown to be present on the cell surface gp55-116 (gB). Utilizing HeLa cells infected with the gp55-116 recombinant vaccinia virus as a specific immunosorbent, we have shown that approximately 40 to 70% of the total serum virus-neutralizing activity of a group of individuals with past HCMV infections was directed against this single envelope glycoprotein. The implications of this finding for vaccine development are discussed.

Human cytomegalovirus contains an envelope glycoprotein of 58 kilodaltons (gp58). The protein, which is derived from a glycosylated precursor molecule of 160 kilodaltons via proteolytic cleavage, is capable of inducing neutralizing antibodies. We have mapped the epitopes recognized by the neutralizing monoclonal antibody 7-17 and a second antibody (27-287) which is not neutralizing. Overlapping fragments of the carboxy-terminal part of the open reading frame coding for gp58 were expressed in Escherichia coli as beta-galactosidase fusion proteins. The reactivities of antibodies 7-17 and 27-287 were determined by Western blot (immunoblot) analysis. Both antibodies recognized sequences between amino acids 608 and 625 of the primary gp58 translation product. The antibodies almost completely inhibited one another in a competitive binding assay with intact virus as antigen. Moreover, antibody 27-287 was able to inhibit the complement-independent neutralizing activity of antibody 7-17.

The processing pathway of the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus was studied using inhibitors of glycosylation and endoglycosidases. The results of these studies indicated that the mature gp55-116 is synthesized by the addition of both simple and complex N-linked sugars to a nonglycosylated precursor of estimated Mr 105,000. In a rapid processing step, the Mr 105,000 precursor is glycosylated to a protein of Mr 150,000 (gplS0) which contains only endoglycosidase H-sensitive sugar linkages. The gpl50 is then processed relatively slowly to a Mr 165,000 to 170,000 species (gpl65-170), which is then cleaved to yield the mature gp55-116. Monensin prevented the final processing steps of the gpl50, including cleavage, suggesting that transport through the Golgi apparatus is required for complete processing. Digestion of the intracellular forms of this complex as well as the virion forms confirmed the above findings and indicated that the mature virion form of gp55 contains 8,000 daltons of N-linked sugars. The virion gpll6 contains some 52,000 to 57,000 daltons of N-linked carbohydrates and approximately 5,000 daltons of 0-linked sugars.

The disulfide-linked glycoprotein B (gB; gp55-116) complex of human cytomegalovirus represents the most abundant and immunogenic component of the virion envelope. We have studied the oligomerization and transport of this molecule, using a series of murine monoclonal antibodies. Our results indicated that oligomerization of this molecule occurred shortly after its synthesis, with a half-time of maximal formation of approximately 25 min. The oligomeric form had an estimated mass of 340,000 Da and likely consisted of a homodimer of the gp55-116 complex. By using a conformation-specific monoclonal antibody, postoligomerization folding of this molecule was demonstrated. This event exhibited an unusually prolonged half-maximal time of approximately 160 min. Both oligomerization and folding occurred in the endoplasmic reticulum.