INTRACELLULAR PROCESSING OF PSEUDORABIES VIRUS GLYCOPROTEIN-M (GM) - GM OF STRAIN BARTHA LACKS N-GLYCOSYLATION

Genes encoding homologs of the herpes simplex virus type 1 UL10 produc t, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa s tructural component of purified virions. A gM(-)PrV mutant could be pr opagated in cell culture, albeit at lower titers and with delayed pene tration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection, PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and secon d hydrophobic region of the protein. This N-glycosylation site is cons erved in all gM homologs sequenced so far, indicating an important fun ctional role. To analyze intracellular processing of PrV gM, Western b lot analyses were performed. In PrV-infected cells, mature 45-kDa gM a s well as 33- and 35-kDa precursor forms were detectable. Presumably d imeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as show n by endoglycosidase digestions. Investigation of several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated vir us used as vaccine, was not modified by N-glycosylation. Sequence anal ysis showed that the N-glycosylation consensus sequence was altered fr om NDT to NDA, which resulted in loss of the N-glycosylation signal. T o our knowledge, this is the only gM homolog identified so far which i s not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. Th e resulting recombinant expressed a UL10 protein lacking N-glycans. In vitro replication analyses did not reveal any difference in virus pro duction, but plaque size and penetration kinetics were slightly reduce d. In summary, we show that wild-type gM is modified by N-glycosylatio n at one conserved site. However, although this site is highly conserv ed throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV i n cell culture. (C) 1997 Academic Press.