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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 GP2b (previously named GS), GP3, GP4, and GP5 (previously named GL). Proteins N, M, and GP5 are major virion components, E occurs in virus particles in intermediate amounts, and GP4, GP3, and GP2b are minor structural proteins. The M and GP5 proteins occur in virus particles as disulfide-linked heterodimers while the GP4, GP3, and GP2b 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 GP2b, GP3, or GP4 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 GP2b, GP2b, and GP4 proteins into viral particles. EAV particles lacking GP2b, GP3, GP4, 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 GP2b/GP2b/GP3/GP4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid. [ABSTRACT FROM AUTHOR]

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[sub 5] (previously named G[sub L]) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP[sub 2b] (previously named G[sub S]), GP[sub 3], and GP[sub 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[sub 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[sub 2b], GP[sub 3], and GP[sub 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[sub 2b] protein in the assembly of the GP[sub 2b]/GP[sub 3]/GP[sub 4] complex. Open reading frames encoding cysteine-to-serine mutants of the GP[sub 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[sub 2b] forms an intermolecular cystine bridge with one of the cysteines of the GP[sub 4] protein, while the cysteine residues at positions 48 and 137 of GP[sub 2b] are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP[sub 4] protein is responsible for the covalent association of GP[sub 3] with the disulfide-linked GP[sub 2b]/GP[sub 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. [ABSTRACT FROM AUTHOR]

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[sub 5] (previously named G[sub L]), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP[sub 2b] (formerly designated G[sub S]), GP[sub 3], and GP[sub 4]. Analysis of the appearance of the GP[sub 2b], GP[sub 3], and GP[sub 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[sub 2b] and GP[sub 4] and heterotrimers of GP[sub 2b], GP[sub 3], and GP[sub 4]. Shortly after their release from infected cells, virions contained mainly cystine-linked GP[sub 2b]/GP[sub 4] heterodimers, which were subsequently converted into disulfide-bonded GP[sub 2b]/GP[sub 3]/GP[sub 4] trimers through the covalent recruitment of GP[sub 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[sub 2b]/GP[sub 3]/GP[sub 4] trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP[sub 3] with the cystine-linked GP[sub 2b]/GP[sub 4] dimer in freshly released virions, nor did we observe higher-order structures of the GP[sub 2b]/GP[sub 4] or GP[sub 2b]/GP[sub 3]/GP[sub 4] complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP[sub 2b]/GP[sub 3]/GP[sub 4] heterotrimers at 4°C suggests that the three minor glycoproteins of EAV are assembled as trimeric... [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes. [ABSTRACT FROM AUTHOR]

Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by infection are directed predominantly against an immunodominant region in the membraneproximal domain of the viral envelope glycoprotein GL, allowing recently the establishment of a sensitive peptide enzyme-linked immunosorbent assay (ELISA) based on this particular domain (J. Nugent et al., J. Virol. Methods 90:167-183, 2000). By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-G[sub L]Δ, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-G[sub L]Δ did not induce a measurable response in our G[sub L]peptide ELISA while the challenge infection of the animals clearly did. EAV-G[sub L]Δ or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals. [ABSTRACT FROM AUTHOR]