The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease.

ABSTRACT In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.

Download Full-text

Efficient Homologous RNA Recombination and Requirement for an Open Reading Frame during Replication of Equine Arteritis Virus Defective Interfering RNAs

Journal of Virology ◽

10.1128/jvi.74.19.9062-9070.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9062-9070 ◽

Cited By ~ 21

Author(s):

Richard Molenkamp ◽

Sophie Greve ◽

Willy J. M. Spaan ◽

Eric J. Snijder

Keyword(s):

Rna Virus ◽

Rna Replication ◽

Proximal Region ◽

Full Length ◽

Equine Arteritis Virus ◽

Open Reading Frame ◽

Replicase Gene ◽

Rna Recombination ◽

Reading Frame ◽

A Genome

ABSTRACT Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156–3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3′- and 5′-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5′-proximal region was found to be approximately 100-fold lower than that in the 3′-proximal part of the genome.

Download Full-text

An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene

Journal of General Virology ◽

10.1099/vir.0.82590-0 ◽

2007 ◽

Vol 88 (4) ◽

pp. 1196-1205 ◽

Cited By ~ 22

Author(s):

Erwin van den Born ◽

Clara C. Posthuma ◽

Kèvin Knoops ◽

Eric J. Snijder

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Heterologous Gene Expression ◽

Equine Arteritis Virus ◽

Expression Vectors ◽

Wild Type Virus ◽

Foreign Gene ◽

Replicase Gene ◽

Green Fluorescent

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP–nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.

Download Full-text

Genetic characterization of equine arteritis virus during persistent infection of stallions

Journal of General Virology ◽

10.1099/vir.0.19545-0 ◽

2004 ◽

Vol 85 (2) ◽

pp. 379-390 ◽

Cited By ~ 35

Author(s):

Udeni B. R. Balasuriya ◽

Jodi F. Hedges ◽

Victoria L. Smalley ◽

Andrea Navarrette ◽

William H. McCollum ◽

...

Keyword(s):

Persistent Infection ◽

Reproductive Tract ◽

Genetic Characterization ◽

Neutralization Assay ◽

High Titre ◽

Equine Arteritis Virus ◽

Replicase Gene ◽

Defective Interfering Particles ◽

Genomic Deletions

Equine arteritis virus (EAV) causes a persistent infection of the reproductive tract of carrier stallions. The authors determined the complete genome sequences of viruses (CW96 and CW01) that were present 5 years apart in the semen of a carrier stallion (CW). The CW96 and CW01 viruses respectively had only 85·6 % and 85·7 % nucleotide identity to the published sequence of EAV (EAV030). The CW96 and CW01 viruses had two 1 nt insertions and a single 1 nt deletion in the leader sequence, and a 3 nt coding insertion in ORF1a; thus their genomes included 12 708 nt as compared to the 12 704 nt in EAV030. Variation between viruses present in the semen of stallion CW and EAV030 was especially marked in the replicase gene (ORF1a and 1b), and the greatest variation occurred in the portion of ORF1a encoding the nsp2 protein. The ORFs 3 and 5, which respectively encode the GP3 and GP5 envelope proteins, showed greatest variation amongst ORFs encoding structural EAV proteins. Comparative sequence analyses of CW96 and CW01 indicated that ORFs 1a, 1b and 7 were highly conserved during persistent infection, whereas there was substantial variation in ORFs 3 and 5. Although the variation that occurs in ORF5 results in the emergence of novel phenotypic viral variants as determined by neutralization assay, all variants were neutralized by high-titre polyclonal equine antisera, suggesting that immune evasion is unlikely to be responsible for the establishment of persistent EAV infection of carrier stallions. Northern blot analyses of RNA extracted from cell culture propagated viruses isolated from 10 different persistently infected stallions failed to demonstrate any large genomic deletions, suggesting that defective interfering particles are also unlikely to be important in either the maintenance or clearance of persistent EAV infection of the reproductive tract of carrier stallions.

Download Full-text

Identification of a Novel Structural Protein of Arteriviruses

Journal of Virology ◽

10.1128/jvi.73.8.6335-6345.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6335-6345 ◽

Cited By ~ 116

Author(s):

Eric J. Snijder ◽

Hans van Tol ◽

Ketil W. Pedersen ◽

Martin J. B. Raamsman ◽

Antoine A. F. de Vries

Keyword(s):

Reverse Genetics ◽

Equine Arteritis Virus ◽

Progeny Virus ◽

Replicase Gene ◽

Structural Protein ◽

Reading Frame ◽

Novel Gene ◽

E Protein ◽

C Terminus ◽

Infected Cells

ABSTRACT 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 GS 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 GS proteins are essential for the production of infectious progeny virus.

Download Full-text