Affiliation:
1. Centre de Recherche en Virologie, Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada H7N 4Z3,1 and
2. Institut de Recherche en Biotechnologie, Montréal, Québec, Canada H4P 2R22
Abstract
ABSTRACT
The GP
3
protein of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in 293 cells by a recombinant human type 5 adenovirus carrying the open reading frame 3 gene. The protein exhibited a molecular mass of 42 kDa and comigrated with GP
3
expressed in PRRSV-infected MARC-145 cells. Removal of N-linked glycans from GP
3
resulted in a 27-kDa protein (P3), confirming its highly glycosylated nature. Pulse-chase experiments carried out either in the context of PRRSV infection or upon individual expression of GP
3
in 293 cells showed that the protein remains completely endo-β-
N
-acetylglucosaminidase H-sensitive even after 4 h of synthesis. Thus, the transport of GP
3
was restricted to the premedial Golgi compartment, presumably the endoplasmic reticulum (ER). However, a minor fraction of GP
3
was found to be secreted in the culture medium as a soluble membrane-free form. This released protein (sGP
3
) was readily identified upon individual expression of GP
3
in 293 cells as well as in the context of PRRSV infection, albeit at lower levels. The sGP
3
migrated as a smear and displayed a molecular mass ranging from 43 to 53 kDa. The unglycosylated form of sGP
3
comigrated with its intracellular deglycosylated counterpart, suggesting that the release from the cell of a subset of GP
3
did not result from cleavage of a putative membrane-anchor sequence. Strikingly, unlike GP
3
, the sGP
3
acquired Golgi-specific modifications of its carbohydrate side chains and folded into a disulfide-linked homodimer. Brefeldin A treatment completely abolished the release of sGP
3
, suggesting that the ER-to-Golgi compartment is an obligatory step in cellular secretion of sGP
3
. In contrast, 10 mM monensin did not prevent sGP
3
release but inhibited the terminal glycosylation that confers on the protein its diffuse pattern. Since GP
3
was found to be nonstructural in the case of the North American strain, secretion of a minor fraction of GP
3
might be an explanation for its high degree of immunogenicity in infected pigs. Furthermore, this secreted protein might be relevant as a model for further studies on the cellular subcompartments involved in the sorting of proteins to the extracellular milieu.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Reference60 articles.
1. A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity;Ascadi G.;Hum. Mol. Genet.,1994
2. Post-translational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas;Bole D. G.;J. Cell Biol.,1986
3. Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum;Bonifacino J. S.;EMBO J.,1991
4. The role of envelope proteins in hepatitis B virus assembly;Bruss V.;Proc. Natl. Acad. Sci. USA,1991
5. Nidovirales: a new order comprising Coronaviridae and Arteriviridae;Cavanagh D.;Arch. Virol.,1997