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Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope.
- Source :
-
Virology [Virology] 2000 Apr 25; Vol. 270 (1), pp. 84-97. - Publication Year :
- 2000
-
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.<br /> (Copyright 2000 Academic Press.)
- Subjects :
- 5' Untranslated Regions genetics
Amino Acid Sequence
Animals
Antibodies, Monoclonal immunology
Base Sequence
Cell Line
Cloning, Molecular
Coronavirus M Proteins
Deoxyribonucleases, Type II Site-Specific metabolism
Epitopes immunology
Equartevirus physiology
Genome, Viral
Glycosylation
Molecular Sequence Data
Murine hepatitis virus genetics
Murine hepatitis virus immunology
Mutagenesis, Insertional genetics
Open Reading Frames genetics
RNA, Viral genetics
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins immunology
Recombinant Fusion Proteins metabolism
Viral Matrix Proteins genetics
Viral Matrix Proteins immunology
Viral Matrix Proteins metabolism
Virus Replication
DNA, Complementary genetics
Epitopes genetics
Equartevirus genetics
Genes, Overlapping genetics
Genes, Viral genetics
Genetic Engineering
Subjects
Details
- Language :
- English
- ISSN :
- 0042-6822
- Volume :
- 270
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Virology
- Publication Type :
- Academic Journal
- Accession number :
- 10772982
- Full Text :
- https://doi.org/10.1006/viro.2000.0245