Genetic Manipulation of Equine Arteritis Virus Using Full-Length cDNA Clones: Separation of Overlapping Genes and Expression of a Foreign Epitope

Abstract

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.

Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity

“…The recent development of reverse-genetics systems (16,31,47) has created new avenues to explore the properties and functions of the unique but poorly characterized set of structural proteins that is used by arteriviruses. It has now been established that the products of all seven genes in the 3Ј-proximal region of the genomes of EAV and the swine arterivirus PRRSV can be detected in virus particles (15,33,34,45,48,49,50).…”

“…EAV infectious cDNA clone pEAV030 (47) was the backbone for all mutant constructs used in this study. The previously designed construct pA45 (18), in which the small overlap between EAV ORF4 and ORF5 had been removed (16), was used for sitedirected mutagenesis of the GP 5 ectodomain and residue Cys-8 in the M protein (Tables 1 and 2). The small insertion made to functionally separate ORF4 and ORF5 did not significantly impair virus replication or infectivity and was stable on repeated virus passaging (16).…”

“…The specific roles of the various envelope proteins in virus assembly and infectivity have not yet been established. However, the recent development of infectious cDNA clones for arteriviruses (16,31,47) has opened the possibility of studying arterivirus assembly by modifying the expression and properties of structural proteins. It was shown that the proteins encoded by all seven genes in the 3Ј-terminal region of the EAV genome (ORF2a to ORF7) are required for the production of infectious progeny virus (35).…”

Generation of a Candidate Live Marker Vaccine for Equine Arteritis Virus by Deletion of the Major Virus Neutralization Domain

“…To construct pEANG L ⌬, a PCR product was synthesized with the oligonucleotides 983 and 984 and pEAN515 as the DNA template. The plasmid pEAN515 is a derivative of EAV infectious cDNA clone pEAV030-BglII KO (22,63) in which the cryptic transcription termination signal at position 8941 to 8956 was removed by site-directed mutagenesis and an MscI site was simultaneously created (mutations: A8941C, T8945A, C8946G, T8947C, T8950C, and G8956C). The resulting PCR fragment (724 nucleotides) was treated with BglII and EcoRI, and the 638-bp digestion product was cloned into BglII-and EcoRI-digested pEAN515 and sequenced.…”

“…The resulting PCR fragment (724 nucleotides) was treated with BglII and EcoRI, and the 638-bp digestion product was cloned into BglII-and EcoRI-digested pEAN515 and sequenced. EAV RNA was transcribed in vitro from pEANG L ⌬ and from pEAN515, and parallel cultures of BHK-21 C13 cells were transfected with the synthetic RNAs by electroporation essentially as described by de Vries et al (22). Infections and mock infections of BHK-21 cells with wild-type or mutant EAV at a high multiplicity of infection (MOI) have also been previously reported (71).…”