EFFECT OF CYSTEINE SUBSTITUTIONS ON DIMERIZATION AND INTERFRAGMENT LINKAGE OF HUMAN CYTOMEGALOVIRUS GLYCOPROTEIN-B (GP-UL55)

Experiments were carried out to analyze the function of cysteine resid ues at amino acid positions 506 (cI), 550 (cII), 573 (cIII), and 610 ( cIV), in dimerization and/or disulfide linkage of human cytomegaloviru s (HCMV) glycoprotein B (gB). Single c-codons or pairs were substitute d in the gB sequence of constructs which were used for transfection an d selection of stable transfectants. Analysis of gB expression product s revealed that single substitutions of cIII or cIV, but neither singl e nor double substitutions of cI or/and cII prevented gB dimerization. All substituted gB derivatives were, however, no longer processed by proteolytic cleavage. After deletion of the membrane anchor domain, co rrect proteolytic processing was again observed for anchorless gB form s. Substitutions of cI or cI/cII in secretory gB appeared to interfere with disulfide linkage between gB cleavage fragments. In the case of anchorless gB with substitutions of cII, cIII, or cIII/cIV, however, e xtracellular gB forms were not recovered. Using the Sindbis expression system recovery of all anchorless gB forms with cysteine substitution s was achieved. Analysis verified involvement of cI/VII substitutions in intrachain disulfide linkage between cleavage fragments of HCMV gB.