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The 5(-terminal leader sequence of the equine arteritis virus (EAV) genome contains an open reading frame (ORF) with an AUG codon in a suboptimal context for initiation of protein synthesis. To investigate the significance of this intraleader ORF (ILO), an expression plasmid was generated carrying a DNA copy of the subgenomic mRNA7 behind a T7 promoter. Capped RNA transcribed from this construct was shown to direct, in an in vitro translation system, the synthesis of leader peptide as well as N protein. Site-directed mutations aimed to either optimize or weaken the sequence context of the ILO start codon affected leader peptide synthesis as predicted; no peptide was detected when the initiation codon was incapacitated. Translation of the downstream N gene was inversely affected by leader peptide production, consistent with a ribosomal leaky scanning mechanism. To investigate the role of the leader peptide in the EAV replication life cycle we generated, using an infectious EAV cDNA clone, two mutant viruses in one of which the ILO start codon was in an optimal Kozak context for translation initiation while in the other the codon was again incapacitated. Surprisingly, both mutant viruses were equally viable and exhibited similar phenotypes in BHK-21 cells. However, their replication kinetics and viral yields were reduced relative to that of the wild-type parental virus, as were their plaque sizes. Importantly, the mutations introduced into the viruses appeared to be rapidly and precisely repaired upon passaging. Already after one viral passage a significant fraction of the viruses had regained the wild-type sequence as well as its phenotype. The results demonstrate that EAV replication is not dependent on the synthesis of the intraleader peptide. Rather, the leader peptide does not seem to have any function in the EAV life cycle. As we discuss, the available data indicate that the ILO 5( nucleotide sequence per se, not its functioning in translation initiation, is of critical importance for EAV replication.

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 membrane-proximal domain of the viral envelope glycoprotein G L , 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 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 L Δ did not induce a measurable response in our G L -peptide ELISA while the challenge infection of the animals clearly did. EAV-G L Δ or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.

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 disulfide-bonded 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 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) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3′-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material.

Equine arteritis virus (EAV) is the prototypic member of the family Arteriviridae, which together with the Corona- and Toroviridae constitutes the order Nidovirales. A common trait of these positive-stranded RNA viruses is the 3′-coterminal nested set of six to eight leader-containing subgenomic mRNAs which are generated by a discontinuous transcription mechanism and from which the viral open reading frames downstream of the polymerase gene are expressed. In this study, we investigated whether the unique gene expression strategy of the Nidovirales could be utilized to convert them into viral expression vectors by introduction of an additional transcription unit into the EAV genome directing the synthesis of an extra subgenomic mRNA. To this end, an expression cassette consisting of the gene for a green fluorescent protein (GFP) flanked at its 3′ end by EAV-specific transcription-regulating sequences was constructed. This genetic module was inserted into the recently obtained mutant infectious EAV cDNA clone pBRNX1.38-5/6 (A. A. F. de Vries, et al., 2000, Virology 270, 84–97) between the genes for the M and the GL proteins. Confocal fluorescence microscopy of BHK-21 cells electroporated with capped RNA transcripts derived from the resulting plasmid (pBRNX1.38-5/6-GFP) demonstrated that the GFP gene was expressed in the transfected cells, while the gradual spread of the infection through the cell monolayer showed that the recombinant virus was replication competent. The development of the cytopathic effect was, however, much slower than in cells that had received equivalent amounts of pBRNX1.38-5/6 RNA, indicating that the vector virus had a clear growth disadvantage compared to its direct precursor. Immunoprecipitation analyses of proteins from metabolically labeled BHK-21 cells infected with supernatant of the transfected cultures confirmed that the recombinant virus vector was viable and expressed viral genes as well as the GFP gene. Reverse transcription-PCR of the viral mRNAs extracted from cells infected with the vector virus revealed that it directed the synthesis of nine instead of eight different EAV RNAs. These findings were corroborated by hybridization analyses. Mapping of the leader-to-body junctions of the ninth mRNA indicated that the 3′ part of the GFP gene contains cryptic transcription signals which gave rise to at least five different RNA species ranging in size from 1277 to 1439 nt [without oligo(A) tract]. Furthermore, translation of the unintended mRNA resulted in the production of an extended version of the EAV M protein. Serial passage of the recombinant virus vector led to its gradual replacement by viral mutants carrying deletions in the GFP gene. The reduction in viral fitness associated with the insertion of the expression cassette into the EAV genome apparently caused genetic instability of the recombinant virus.

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5′ part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3′ part of EAV ORF 2a overlaps with the 5′ part of the former ORF 2 (now renamed ORF 2b), which encodes the G S glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and G S proteins are essential for the production of infectious progeny virus.

Equine arteritis virus (EAV) belongs to a group of small enveloped positive-stranded RNA viruses, provisionally designated arteriviruses, which further includes lactate dehydrogenase-elevating virus (LDV), simian hemorrhagic fever virus (SHFV), and swine infertility and respiratory syndrome virus (SIRSV) or Lelystad virus (LV). These viruses resemble togaviruses in their physicochemical properties, the polarity and size (13–15 kb) of their genomes, and the size (50–70 nm) and morphology (isometric) of the virionsl,2 but virus assembly occurs by intracellular budding1,3. In contrast, the genome organization and gene expression strategy are similar to those of corona-and toroviruses2,4,5,6,7,8.

Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonsegmented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the two families. Here, the common traits and distinguishing features of the Nidovirales are reviewed.