A subset of porcine reproductive and respiratory syndrome virus GP3 glycoprotein is released into the culture medium of cells as a non-virion-associated and membrane-free (soluble) form

The GP, 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, in 293 cells showed that the protein remains completely endo-beta-N-acetylglucosaminidase H-sensitive even after 3 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, 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.