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 GP3 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 GP3 expressed in PRRSV-infected MARC-145 cells. Removal of N-linked glycans from GP3resulted 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 GP3 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 GP3 was restricted to the premedial Golgi compartment, presumably the endoplasmic reticulum (ER). However, a minor fraction of GP3 was found to be secreted in the culture medium as a soluble membrane-free form. This released protein (sGP3) was readily identified upon individual expression of GP3 in 293 cells as well as in the context of PRRSV infection, albeit at lower levels. The sGP3 migrated as a smear and displayed a molecular mass ranging from 43 to 53 kDa. The unglycosylated form of sGP3 comigrated with its intracellular deglycosylated counterpart, suggesting that the release from the cell of a subset of GP3 did not result from cleavage of a putative membrane-anchor sequence. Strikingly, unlike GP3, the sGP3 acquired Golgi-specific modifications of its carbohydrate side chains and folded into a disulfide-linked homodimer. Brefeldin A treatment completely abolished the release of sGP3, suggesting that the ER-to-Golgi compartment is an obligatory step in cellular secretion of sGP3. In contrast, 10 mM monensin did not prevent sGP3 release but inhibited the terminal glycosylation that confers on the protein its diffuse pattern. Since GP3 was found to be nonstructural in the case of the North American strain, secretion of a minor fraction of GP3 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.

The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163

“…GP5 is the most abundant glycoprotein found on the surface of the virion and hence is named the major envelope glycoprotein, whereas the GP2a, GP3, and GP4 proteins, which are also present on the surface of the virion in less abundant quantities, are termed the minor envelope glycoproteins. Although early studies reported that the GP3 protein is not a structural component of the North American (genotype II) PRRS virions (24,34), recent studies from our laboratory have shown that it is present on the virion envelope (10), an observation consistent with the European (genotype I) PRRS virions (49). All of the major and minor envelope proteins are required for generation of infectious PRRSV (53).…”

Characterization of Two New Structural Glycoproteins, GP 3 and GP 4 , of Equine Arteritis Virus

“…In contrast, for the PRRSV IAF-Klop strain, the GP 3 protein was characterized as nonstructural. Upon individual expression, as well as in the context of a PRRSV infection, a small fraction of the IAF-Klop GP 3 protein was shown to be secreted into the extracellular medium but the protein could not be identified in virions (20,31). For LDV, in vitro transcription and translation studies revealed that the ORF3-encoded protein was soluble or weakly associated with membranes through an uncleaved signal peptide.…”

“…In the Lelystad strain of PRRSV, it was found that their products (designated GP 4 and GP 3 , respectively) are minor glycoproteins of the virus (47). In contrast, the ORF3-encoded protein of the Quebec strain of PRRSV (IAF-Klop) has been shown to code for a soluble nonstructural protein (20,31). In vitro translation experiments showed that the ORF3-encoded protein of LDV is also a soluble protein (14).…”

The structural biology of PRRSV

“…Some GP3 protein is secreted into the cell medium during PRRSV type 2 infections Mardassi et al, 1998;Wierenga et al, 2002) and it was initially thought not to be a structural protein. However, GP3 was subsequently identified as a minor component in both type 1 and 2 viruses (de Lima et al, 2009;van Nieuwstadt et al, 1996;Wissink et al, 2005).…”