FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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.
Get Full Text
Publication Date: 2000-04-25 PubMed ID: 10772982DOI: 10.1006/viro.2000.0245Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • 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

              Citations

              This article has been cited 22 times.
              1. Matczuk AK, Chodaczek G, Ugorski M. Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells. Viruses 2019 Aug 9;11(8).
                doi: 10.3390/v11080735pubmed: 31404947google scholar: lookup
              2. Di H, Morantz EK, Sadhwani H, Madden JC Jr, Brinton MA. Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV). Virology 2018 Dec;525:150-160.
                doi: 10.1016/j.virol.2018.09.014pubmed: 30286427google scholar: lookup
              3. Wang C, Meng H, Gao Y, Gao H, Guo K, Almazan F, Sola I, Enjuanes L, Zhang Y, Abrahamyan L. Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus. Vet Res 2017 Aug 10;48(1):41.
                doi: 10.1186/s13567-017-0445-2pubmed: 28797297google scholar: lookup
              4. Han M, Yoo D. Engineering the PRRS virus genome: updates and perspectives. Vet Microbiol 2014 Dec 5;174(3-4):279-295.
                doi: 10.1016/j.vetmic.2014.10.007pubmed: 25458419google scholar: lookup
              5. Balasuriya UB, Zhang J, Go YY, MacLachlan NJ. Experiences with infectious cDNA clones of equine arteritis virus: lessons learned and insights gained. Virology 2014 Aug;462-463:388-403.
                doi: 10.1016/j.virol.2014.04.029pubmed: 24913633google scholar: lookup
              6. Wang C, Huang B, Kong N, Li Q, Ma Y, Li Z, Gao J, Zhang C, Wang X, Liang C, Dang L, Xiao S, Mu Y, Zhao Q, Sun Y, Almazan F, Enjuanes L, Zhou EM. A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit. Vet Res 2013 Oct 31;44(1):104.
                doi: 10.1186/1297-9716-44-104pubmed: 24176053google scholar: lookup
              7. Balasuriya UB, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013 Nov 29;167(1-2):93-122.
                doi: 10.1016/j.vetmic.2013.06.015pubmed: 23891306google scholar: lookup
              8. Tian D, Wei Z, Zevenhoven-Dobbe JC, Liu R, Tong G, Snijder EJ, Yuan S. Arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture. J Virol 2012 Apr;86(7):3701-12.
                doi: 10.1128/JVI.06836-11pubmed: 22258262google scholar: lookup
              9. Firth AE, Zevenhoven-Dobbe JC, Wills NM, Go YY, Balasuriya UBR, Atkins JF, Snijder EJ, Posthuma CC. Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production. J Gen Virol 2011 May;92(Pt 5):1097-1106.
                doi: 10.1099/vir.0.029264-0pubmed: 21307223google scholar: lookup
              10. Yu D, Lv J, Sun Z, Zheng H, Lu J, Yuan S. Reverse genetic manipulation of the overlapping coding regions for structural proteins of the type II porcine reproductive and respiratory syndrome virus. Virology 2009 Jan 5;383(1):22-31.
                doi: 10.1016/j.virol.2008.09.013pubmed: 18977502google scholar: lookup
              11. Choi YJ, Yun SI, Kang SY, Lee YM. Identification of 5' and 3' cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5' AU-rich sequences restored replication of a 5'-proximal 7-nucleotide deletion mutant. J Virol 2006 Jan;80(2):723-36.
                doi: 10.1128/JVI.80.2.723-736.2006pubmed: 16378975google scholar: lookup
              12. de Haan CA, Haijema BJ, Boss D, Heuts FW, Rottier PJ. Coronaviruses as vectors: stability of foreign gene expression. J Virol 2005 Oct;79(20):12742-51.
                doi: 10.1128/JVI.79.20.12742-12751.2005pubmed: 16188977google scholar: lookup
              13. Enjuanes L, Sola I, Alonso S, Escors D, Zúñiga S. Coronavirus reverse genetics and development of vectors for gene expression. Curr Top Microbiol Immunol 2005;287:161-97.
                doi: 10.1007/3-540-26765-4_6pubmed: 15609512google scholar: lookup
              14. Yoo D, Welch SK, Lee C, Calvert JG. Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors. Vet Immunol Immunopathol 2004 Dec 8;102(3):143-54.
                doi: 10.1016/j.vetimm.2004.09.019pubmed: 15507301google scholar: lookup
              15. Takahashi-Omoe H, Omoe K, Sakaguchi M, Kameoka Y, Matsushita S, Inada T. Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins. Comp Immunol Microbiol Infect Dis 2004 Mar;27(2):81-92.
                doi: 10.1016/S0147-9571(03)00053-5pubmed: 14690718google scholar: lookup
              16. Castillo-Olivares J, Wieringa R, Bakonyi T, de Vries AA, Davis-Poynter NJ, Rottier PJ. Generation of a candidate live marker vaccine for equine arteritis virus by deletion of the major virus neutralization domain. J Virol 2003 Aug;77(15):8470-80.
                doi: 10.1128/jvi.77.15.8470-8480.2003pubmed: 12857916google scholar: lookup
              17. Ortego J, Sola I, Almazán F, Ceriani JE, Riquelme C, Balasch M, Plana J, Enjuanes L. Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence. Virology 2003 Mar 30;308(1):13-22.
                doi: 10.1016/s0042-6822(02)00096-xpubmed: 12706086google scholar: lookup
              18. Snijder EJ, Dobbe JC, Spaan WJ. Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity. J Virol 2003 Jan;77(1):97-104.
                doi: 10.1128/jvi.77.1.97-104.2003pubmed: 12477814google scholar: lookup
              19. de Haan CA, Volders H, Koetzner CA, Masters PS, Rottier PJ. Coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization. J Virol 2002 Dec;76(24):12491-502.
                doi: 10.1128/jvi.76.24.12491-12502.2002pubmed: 12438575google scholar: lookup
              20. Bautista EM, Faaberg KS, Mickelson D, McGruder ED. Functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus. Virology 2002 Jul 5;298(2):258-70.
                doi: 10.1006/viro.2002.1495pubmed: 12127789google scholar: lookup
              21. Enjuanes L, Sola I, Almazan F, Ortego J, Izeta A, Gonzalez JM, Alonso S, Sanchez JM, Escors D, Calvo E, Riquelme C, Sanchez C. Coronavirus derived expression systems. J Biotechnol 2001 Jul 12;88(3):183-204.
                doi: 10.1016/s0168-1656(01)00281-4pubmed: 11434966google scholar: lookup
              22. Pasternak AO, Gultyaev AP, Spaan WJ, Snijder EJ. Genetic manipulation of arterivirus alternative mRNA leader-body junction sites reveals tight regulation of structural protein expression. J Virol 2000 Dec;74(24):11642-53.
                doi: 10.1128/jvi.74.24.11642-11653.2000pubmed: 11090163google scholar: lookup
              Subscribe to our newsletter
              Join 99,727 horse owners like you who receive our email updates on horse health and nutrition.