Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP 2b (previously named G S ), GP 3 , GP 4 , and GP 5 (previously named G L ). Proteins N, M, and GP 5 are major virion components, E occurs in virus particles in intermediate amounts, and GP 4 , GP 3 , and GP 2b are minor structural proteins. The M and GP 5 proteins occur in virus particles as disulfide-linked heterodimers while the GP 4 , GP 3 , and GP 2b proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP 2b , GP 3 , or GP 4 proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP 2b , GP 3 , and GP 4 proteins into viral particles. EAV particles lacking GP 2b , GP 3 , GP 4 , and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP 2b /GP 3 /GP 4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.Equine arteritis virus (EAV) is the prototypic member of the Arteriviridae family (order Nidovirales), which also includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian hemorrhagic fever virus (4). Virus particles have a diameter of approximately 60 nm and consist of a 12.7-kb RNA genome of positive polarity that is packaged by the 14-kDa nucleocapsid protein (N) into a putatively icosahedral core, which is surrounded by a lipid-containing envelope with small surface projections (17,20).In the EAV envelope six viral proteins have been identified (6, 27, 43). The 16-kDa unglycosylated membrane protein M and the heterogeneously glycosylated GP 5 (previously named G L ) protein of 30 to 42 kDa are the most abundant envelope proteins and occur in virions as covalently linked heterodimers (7, 26). The membrane topology of the EAV M protein is unknown but its hydropathy profile resembles that of the LDV M protein. The latter protein was previously shown to be a triple-spanning membrane protein having its amino terminus at the outside of the virion and its carboxy terminus at the inside (12). The EAV M protein is, therefore, assumed to contain three internal transmembrane segments...

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...

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP 5 (previously named G L ), which occur as disulfidebonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP 2b (formerly designated G S ), GP 3 , and GP 4 . Analysis of the appearance of the GP 2b , GP 3 , and GP 4 proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP 2b and GP 4 and heterotrimers of GP 2b , GP 3 , and GP 4 . Shortly after their release from infected cells, virions contained mainly cystine-linked GP 2b /GP 4 heterodimers, which were subsequently converted into disulfide-bonded GP 2b /GP 3 /GP 4 trimers through the covalent recruitment of GP 3 . This process occurred faster at a higher pH but was arrested at 4°C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP 2b /GP 3 /GP 4 trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP 3 with the cystine-linked GP 2b /GP 4 dimer in freshly released virions, nor did we observe higher-order structures of the GP 2b /GP 4 or GP 2b /GP 3 /GP 4 complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP 2b /GP 3 /GP 4 heterotrimers at 4°C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP 2b /GP 4 dimer and GP 3 was also inferred from coexpression experiments showing that the presence of GP 3 increased the electrophoretic mobility of the disulfide-bonded GP 2b / GP 4 dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.Equine arteritis virus (EAV) is a single-stranded RNA virus which belongs to the genus Arterivirus. Other members of this single genus in the family Arteriviridae are lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian hemorrhagic fever virus. Despite marked differences in their biophysical properties, genome sizes, and structural protein compositions, the Arteriviridae were joined together with the Coronaviridae and the recently established family Roniviridae in the order Nidovirales on the basis of similarities in their genomic organizations and replication strategies.EAV virions have a diameter of 40 to 60 nm and consist of a putatively icosahedral core that is surrounded by a lipidcontaining envelope with sm...

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP 5 (previously named G L ) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP 2b (previously named G S ), GP 3 , and GP 4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP 5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP 2b , GP 3 , and GP 4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP 2b protein in the assembly of the GP 2b /GP 3 /GP 4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP 2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP 2b forms an intermolecular cystine bridge with one of the cysteines of the GP 4 protein, while the cysteine residues at positions 48 and 137 of GP 2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP 4 protein is responsible for the covalent association of GP 3 with the disulfide-linked GP 2b /GP 4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus which belongs to the genus Arterivirus. Other members of this single genus in the family Arteriviridae are Lactate dehydrogenase-elevating virus (LDV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Simian hemorrhagic fever virus. On the basis of conserved amino acid sequence motifs in their polymerase (poly)proteins and similarities in their genome organization and gene expression strategy, the Arteriviridae have been grouped together with the Coronaviridae and the recently established family Roniviridae in the order Nidovirales (2, 3, 4).The genome of EAV contains nine functional open reading frames (ORFs). ORF1a and -1b occupy the 5Ј three-fourths of the genome and code for proteins involved in viral replication and transcription (6,25,29,30,32). The remaining one-fourth of the viral genome contains seven relatively small ORFs (ORF2a, -2b, and -3 through -7), which are expressed from a 3Ј-coterminal nested set of six leader-containing subgenomic mRNAs (RNA2 through -7) (8, 26) and code for the known viral structur...

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