Structural Protein Requirements in Equine Arteritis Virus Assembly

Abstract

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...

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

“…Envelope protein E is a small, unglycosylated hydrophobic protein necessary for the correct assembly of the EAV virion (23). It was previously demonstrated (4) that ORF2a, encoding protein E, was one of the most conserved genes during persistent infection with only 3 synonymous substitutions found in the course of a 5-year observation.…”

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

“…For EAV, GP 2b , GP 3 , and GP 4 have been reported to exist as covalently linked heterotrimeric complexes (45). Furthermore, incorporation of these glycoproteins and of the E protein in EAV particles was shown to be interdependent (43). The GP 5 and M proteins exist as disulfide-linked heterodimers in PRRSV particles (20).…”

Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity

“…Using an European PRRSV isolate, Wissink et al (2004) have described a heterotrimeric complex of GP2, GP3 and GP4 and, without evidence, speculated a possible incorporation of E protein with this trimeric complex. Wieringa et al (2004) have recently shown a covalent association of the E protein with the GP2b-GP3-GP4 heterotrimers in EAV, suggesting a possible role of the heteromultimeric complex in the virus entry process. It is noteworthy that E proteins of European PRRSV and EAV contain a single cysteine at positions 51 and 58, respectively, which corresponds to position 54 in the North American PRRSV genotype (Fig.…”