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

Abstract

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and G L , the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein G S . In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP 3 and GP 4 , respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP 3 and GP 4 proteins synthesized in EAV-infected cells. The results showed that GP 4 is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP 3 , on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP 3 localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP 3 and GP 4 proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.Equine arteritis virus (EAV), the etiological agent of equine viral arteritis (9,11,45), has been assigned to the family Arteriviridae. The Arteriviridae constitute the single genus Arterivirus. Other members of this genus are lactate dehydrogenaseelevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian hemorrhagic fever virus (SHFV). Although their physicochemical properties, genome sizes, and virion architectures suggest otherwise, on the basis of similarities in genomic organization and replication strategy, the Arteriviridae were grouped together with the Coronaviridae in the order Nidovirales (2, 3, 6, 41).The EAV genome consists of a single, positive-stranded RNA molecule of 12.7 kb that is 5Ј capped and 3Ј polyadenylated (3). The 5Ј three-quarters of the genome contains two open reading frames (ORFs), ORF1a and -1b, that encode the proteins involved in viral RNA replication and transcription (3). Downstream of these ORFs, the genome contains a set of seven smaller ORFs (ORF2a, -2b, and -3 through -7) that are expressed from a 3Ј-cotermi...

Envelope Protein Requirements for the Assembly of Infectious Virions of Porcine Reproductive and Respiratory Syndrome Virus

“…For LDV, the GP 3 protein was reported to be a nonstructural, soluble glycoprotein that is secreted from infected cells (9). In contrast, the GP 3 protein of EAV was clearly demonstrated to be incorporated into virions (44). For PRRSV, both situations have been described.…”

“…While its membrane topology has not been resolved and remains speculative (12,44), its virion structural nature is also still controversial. For LDV, the GP 3 protein was reported to be a nonstructural, soluble glycoprotein that is secreted from infected cells (9).…”

Sequence analysis of minor protein genes of equine arteritis virus during persistent infection

“…The 2 most 5'-proximal ORFs (1a and 1b) occupy approximately three-quarters of the genome and encode 2 replicase polyproteins. The remaining 3' quarter contains eight ORFs which encode 8 structural proteins: E (envelope protein), GP2 (glycoprotein 2), GP3 (glycoprotein 3), GP4 (glycoprotein 4), 5a, GP5 (glycoprotein 5), M (matrix protein), and N (nucleocapsid protein) (8,10,22).…”

Myristoylation of the arterivirus E protein: the fatty acid modification is not essential for membrane association but contributes significantly to virus infectivity

“…4b). Between GP5 and M, various bands were visible representing the (heterogeneously glycosylated) minor proteins GP2b, GP3 and GP4 (Veit et al, 2008a;Wieringa et al, 2002). The E protein could not be identified unambiguously, probably because the mature protein contains only a single methionine.…”