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Home / Equine Research Bank / J Virol / Cloning and characterization of cDNAs encoding equine infect...
Journal of virology1990; 64(8); 3716-3725; doi: 10.1128/JVI.64.8.3716-3725.1990

Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.

Abstract: We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.
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Publication Date: 1990-08-01 PubMed ID: 2164593PubMed Central: PMC249666DOI: 10.1128/JVI.64.8.3716-3725.1990Google Scholar: Lookup
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  • Journal Article
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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 focuses on the isolation and characterization of six cDNA clones from a cell line infected with equine infectious anemia virus (EIAV). The EIAV-infected cell line exhibits a Rev-defective phenotype. The study provides insights into the structure of cDNAs, predicting a bicistronic message wherein Tat is encoded by exons 1 and 2 and the presumed Rev protein is encoded by exons 3 and 4.

Extraction and Characterization of cDNA Clones

  • The researchers extracted six cDNA clones from a cell line infected with Equine Infectious Anemia Virus (EIAV). EIAV is a lentivirus that causes often-fatal anemia in horses.
  • These clones are characterized as having a “Rev-defective” phenotype, indicating that the Rev protein, essential for viral replication, is defective or non-functional.

Understanding cDNA Clones Structure

  • All six clones exhibited a nearly identical splicing pattern, comprised of four exons, units of RNA that contain information for protein coding.
  • The different exons contained different portions of the virus genome, with one containing the Tat gene from mid-genome, and another containing the putative Rev protein from the 3′ end.

The Bicistronic Message

  • The structure of the cDNAs predicts a bicistronic message, a specific type of gene expression in which two proteins are encoded within the same mRNA molecule.
  • The Tat protein, a protein essential for EIAV activation, appears to be encoded by the first two exons, while the defective Rev protein is encoded by exons 3 and 4.

Significance of Tat and Rev Proteins

  • Tat protein’s adequate functionality was confirmed through transient transfections with cDNA expression plasmids.
  • The defective Rev protein manifests as a result of a frameshift in exon 4, leading to a truncated protein and establishing a plausible explanation for the cells’ Rev-defective phenotype.

Confirmation of Findings

  • The researchers used peptide antisera to identify the faulty Rev protein, thus validating the cDNA sequence.
  • They were also able to identify the normal Rev protein in productively infected cells, proving the existence of both functional and defective instances of this protein in the EIAV infection process.

Cite This Article

APA
Stephens RM, Derse D, Rice NR. (1990). Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins. J Virol, 64(8), 3716-3725. https://doi.org/10.1128/JVI.64.8.3716-3725.1990

Publication

Journal of virology
ISSN: 0022-538X
NlmUniqueID: 0113724
Country: United States
Language: English
Volume: 64
Issue: 8
Pages: 3716-3725

Researcher Affiliations

Stephens, R M
  • Laboratory of Molecular Virology and Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21701.
Derse, D
    Rice, N R

      MeSH Terms

      • Amino Acid Sequence
      • Animals
      • Base Sequence
      • Cells, Cultured
      • Cloning, Molecular
      • DNA, Viral / genetics
      • Exons
      • Gene Library
      • Gene Products, rev / genetics
      • Gene Products, tat / genetics
      • Horses
      • Immune Sera
      • Infectious Anemia Virus, Equine / genetics
      • Kidney
      • Molecular Sequence Data
      • Mutation
      • Peptides / chemical synthesis
      • Protein Biosynthesis
      • RNA Splicing
      • RNA, Viral / isolation & purification
      • Sequence Homology, Nucleic Acid
      • Trans-Activators / genetics
      • Transcription, Genetic
      • Transcriptional Activation
      • Transfection

      Grant Funding

      • N01-CO-74101 / NCI NIH HHS

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