THE ROLE OF SITE-SPECIFIC N-GLYCOSYLATION IN SECRETION OF SOLUBLE FORMS OF RABIES VIRUS GLYCOPROTEIN

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotei n is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infect ion. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of th ese sequons is important in the expression of the glycoprotein. Solubl e forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain, Using site-dir ected mutagenesis and expression in transfected eukaryotic cells, it w as possible to compare the effects of site-specific glycosylation on t he cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein, These studies yielded t he surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, onl y the N-glycan at Asn319 permitted secretion of soluble rabies virus g lycoprotein, Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-leng th transmembrane form of rabies virus glycoprotein which contains hete rogeneous oligosaccharides, The current studies demonstrate that a sol uble form of rabies virus glycoprotein containing only one sequon at A sn319 is efficiently secreted in the presence of the N-glycan processi ng inhibitor 1-deoxymannojirimycin, Thus, it is possible to purify a c onformationally relevant form of rabies virus glycoprotein that contai ns only one N-glycan with a substantial reduction in its microheteroge neity, This form of the glycoprotein may be particularly useful for fu ture studies aimed at elucidating the three-dimensional structure of t his important glycoprotein.