A Subset of Porcine Reproductive and Respiratory Syndrome Virus GP 3 Glycoprotein Is Released into the Culture Medium of Cells as a Non-Virion-Associated and Membrane-Free (Soluble) Form

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.