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Journal of virology1994; 68(9); 5755-5764; doi: 10.1128/JVI.68.9.5755-5764.1994

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus.

Abstract: To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.
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Publication Date: 1994-09-01 PubMed ID: 8057457PubMed Central: PMC236979DOI: 10.1128/JVI.68.9.5755-5764.1994Google Scholar: Lookup
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  • Journal Article

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.

The research examines the proteolytic processing of the equine arteritis virus (EAV) using specific antisera. This process was studied in infected cells through methods such as Western blot and immunoprecipitation techniques. Findings discovered that the ORF1a protein underwent at least five proteolytic cleavages, each resulting in proteins of various sizes.

Research Methodologies

For this research, scientists used several methods:

  • Raised specific antisera in rabbits using six synthetic peptides and a bacterial fusion protein as antigens to study the ORF1a protein of the equine arteritis virus (EAV).
  • The processing of the EAV ORF1a protein in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Both methods are experimental procedures used to detect specific proteins based on their ability to bind to specific antibodies.
  • Additional information was gleaned from the transient expression of ORF1a cDNA constructs. This involves introducing the ORF1a gene into a host organism and studying its expression or functionality within that organism.

Findings

The research led to several key findings:

  • The 187-kDa ORF1a protein underwent at least five proteolytic cleavages. Proteolytic cleavage refers to the process where proteins are broken down to smaller pieces.
  • The cleavage products, named nonstructural proteins (nsps) 1 through 6, had weights approximately 29, 61, 22, 31, 41, and 3 kDa. These various sized proteins would have different functional roles in the replication and infection process of the virus.
  • Pulse-chase experiments showed the most rapid processing steps were at the nsp1/2 and nsp2/3 junctions. Pulse-chase experiments are a method used to study the rate of protein synthesis and degradation.
  • The other processing steps were performed by the EAV serine protease in nsp4 and by a third unidentified protease. Proteases are enzymes that break down proteins.

Implication of Findings

These findings provide important insights into the proteolytic processing of the EAV ORF1a protein. Understanding these processes could be key to developing treatments or vaccines for diseases caused by the equine arteritis virus.

Cite This Article

APA
Snijder EJ, Wassenaar AL, Spaan WJ. (1994). Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J Virol, 68(9), 5755-5764. https://doi.org/10.1128/JVI.68.9.5755-5764.1994

Publication

Journal of virology
ISSN: 0022-538X
NlmUniqueID: 0113724
Country: United States
Language: English
Volume: 68
Issue: 9
Pages: 5755-5764

Researcher Affiliations

Snijder, E J
  • Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
Wassenaar, A L
    Spaan, W J

      MeSH Terms

      • Amino Acid Sequence
      • Equartevirus / enzymology
      • Genes, Viral
      • Molecular Sequence Data
      • Open Reading Frames
      • Peptides / chemistry
      • Peptides / immunology
      • Precipitin Tests
      • Protein Processing, Post-Translational
      • RNA-Dependent RNA Polymerase / metabolism
      • Solubility
      • Time Factors
      • Viral Proteins / chemistry
      • Viral Proteins / immunology
      • Viral Proteins / metabolism
      • Viral Structural Proteins / genetics

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