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Virology2000; 270(1); 84-97; doi: 10.1006/viro.2000.0245

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.
Copyright 2000 Academic Press.
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Publication Date: 2000-04-25 PubMed ID: 10772982DOI: 10.1006/viro.2000.0245Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research studied the equine arteritis virus (EAV), with a focus on understanding how its overlapping genes function. Through genetic manipulation, they discovered that it was possible to separate these genes and still maintain the viability of the virus. The researchers also successfully introduced a foreign epitope, a part of the antigen recognized by the immune system, into the virus.

Equine Arteritis Virus and its Genetic Structure

  • The Equine arteritis virus (EAV) is a type of RNA virus from the Arteriviridae family.
  • Its genome is unsegmented and infectious, measuring 12,704 nucleotides in length without the poly(A) tail.
  • The genome contains eight overlapping genes, which creates a ‘nested set’ of mRNAs.

Genetic Manipulation of the Virus

  • The researchers constructed a series of mutant full-length cDNA clones in order to study the importance of gene overlapping in the viral lifecycle.
  • The open reading frames (ORFs) 4 and 5, ORFs 5 and 6, or ORFs 4, 5, and 6 were separated using the enzyme AflII.
  • The RNA transcribed from these modified plasmids was infectious, proving that overlapping gene organization is not necessary for EAV’s viability.
  • Moreover, the mutated viruses were able to replicate almost as efficiently as the wildtype virus and maintain the introduced mutations.

Introduction of a Foreign Epitope

  • The AflII site between ORFs 5 and 6 was used to genetically engineer a virus with an extended ectodomain of the ORF 6-encoded M protein.
  • This extension consisted of nine amino acids from a similar protein in the mouse hepatitis virus (MHV).
  • The added nonapeptide contained an O-glycosylation signal and an epitope recognized by an MHV-specific monoclonal antibody.
  • The recombinant virus successfully expressed both the glycosylation signal and the epitope.
  • While the engineered virus showed a growth disadvantage compared to the original, it was able to retain the foreign genetic material even after three rounds of passage, or replication.

Cite This Article

APA
de Vries AA, Glaser AL, Raamsman MJ, de Haan CA, Sarnataro S, Godeke GJ, Rottier PJ. (2000). Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope. Virology, 270(1), 84-97. https://doi.org/10.1006/viro.2000.0245

Publication

Virology
ISSN: 0042-6822
NlmUniqueID: 0110674
Country: United States
Language: English
Volume: 270
Issue: 1
Pages: 84-97

Researcher Affiliations

de Vries, A A
  • Virology Unit, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, Utrecht, 3584 CL, The Netherlands.
Glaser, A L
    Raamsman, M J
      de Haan, C A
        Sarnataro, S
          Godeke, G J
            Rottier, P J

              MeSH Terms

              • 5' Untranslated Regions / genetics
              • Amino Acid Sequence
              • Animals
              • Antibodies, Monoclonal / immunology
              • Base Sequence
              • Cell Line
              • Cloning, Molecular
              • Coronavirus M Proteins
              • DNA, Complementary / genetics
              • Deoxyribonucleases, Type II Site-Specific / metabolism
              • Epitopes / genetics
              • Epitopes / immunology
              • Equartevirus / genetics
              • Equartevirus / physiology
              • Genes, Overlapping / genetics
              • Genes, Viral / genetics
              • Genetic Engineering
              • 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

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