OLIGOMERIZATION AND POSTTRANSLATIONAL PROCESSING OF GLYCOPROTEIN-G OF HUMAN RESPIRATORY SYNCYTIAL VIRUS - ALTERED O-GLYCOSYLATION IN THE PRESENCE OF BREFELDIN-A

The post-translation maturation of the attachment G glycoprotein of human respiratory synctial virus (RSV) was investigated. The G protein formed homo-oligomers which sedimented in sucrose gradients at the same rate as the fusion F protein tetramer. Oligomerization of the G protein was insensitive to carbonylcyanide m-chlorophenylhydrazine, showing that this step occurs in the endoplasmic reticulum prior to O-glycosylation which initiated in the trans-Golgi compartment. The sedimentation of the G protein oligomer was essentially unchanged by the subsequent addition of O-linked sugars. This indicated that their contribution to the M(r) of the G protein is less than that estimated by electrophoretic mobility. It also suggested that O-glycosylation is not an important determinant of G protein oligomerization and, by implication, of polypeptide folding. The G protein is palmitylated. In short labelling pulses, the G protein accumulated as two species of 48K and 50K which contained only N-linked sugars, whose difference in M(r) was due solely to an N-linked sugar, which both assembled into oligomers, but which differed in the rate of subsequent O-glycosylation. The G protein was not detectably O-glycosylated in the presence of monensin, confirming previous work. In the presence of brefeldin A (BFA), it accumulated as a partially O-glycosylated species (BFA-G) of 68K to 78K. But further analysis by chase incubations following BFA-washout, by lectinbinding, and by glycosidase treatment suggested that BFA-G was not a fully authentic processing intermediate. In particular, some of the O-linked side-chains of the BFA-G protein were found to be sialylated. Rather than being a normal step in processing, this sialylation probably was due to altered distribution or activity of sialytransferases during BFA treatment and may have resulted in the premature termination of elongation of some of the O-liked side-chains. Thus, these studies (i) indicate that O-glycosylation of the G protein begins in the trans-Golgi compartment and (ii) suggest that O-glycosylation is completed in as a subsequent compartment, but this latter suggestion is complicated by the evidence that the BFA-G protein is not a fully authentic intermediate. Finally, an additional species of 48K to 60K was detected under standard conditions of infection and was found to differ in several ways from the 48K and 50K precursors described above: it was detected only following long labelling periods, it was detected only following long labelling periods, it was found in monomers rather than oligomers, and it contained a high content of O-linked sugars. Also, antibodies raised against a peptide from the G ectodomain reacted with the 48K, 50K and mature G proteins but did not react with the 48K to 60K species. This indicated that the latter contained differences in conformation or O-glycosylation which masked that part of the ectodomain. The 48K to 60K species was not detected in virions or secreted from infected cells. Thus, it appeared to be a dead-end by-product rather than a true processing intermediate.