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Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

ABSTRACT: The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.

SUBMITTER: den Boon JA

PROVIDER: S-EPMC240924 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

den Boon J A JA Snijder E J EJ Chirnside E D ED de Vries A A AA Horzinek M C MC Spaan W J WJ

Journal of virology 19910601 6

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of co ...[more]

PMID: 1851863

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Project description:UnlabelledPrevious studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from <3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14(+) monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus.ImportanceOutbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a critical role in EVA pathogenesis. To this point, however, the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of the cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV-susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

Project description: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.

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Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

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Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

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