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Journal of virology2000; 74(7); 3156-3165; doi: 10.1128/jvi.74.7.3156-3165.2000

Isolation and characterization of an arterivirus defective interfering RNA genome.

Abstract: Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.
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Publication Date: 2000-03-09 PubMed ID: 10708432PubMed Central: PMC111816DOI: 10.1128/jvi.74.7.3156-3165.2000Google Scholar: Lookup
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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 paper presents the first documented arterivirus defective interfering (DI) RNAs produced from the Equine arteritis virus (EAV). The findings include the isolation of a DI RNA genome, its sequencing, and further investigation into understanding the sequences required for replication and packaging of EAV DI RNAs.

Generation and Identification of Arterivirus DI RNAs

  • EAV, a single-stranded RNA virus and member of the Arteriviridae family, is known to use a mechanism known as discontinuous transcription to make its nested set of six subgenomic mRNAs, from which its structural genes express.
  • For the first time, a defective interfering (DI) RNA of the arterivirus type is generated. This was done via the method of sequential undiluted passage of wild-type EAV stock in BHK-21 cells.
  • The smallest DI RNA genome, a length of around 5.6 kb, was cloned to produce a copy of cDNA.

Transfection and Segmentation of the DI RNA Genome

  • The cDNA copied DI RNA was transfected into EAV-infected BHK-21 cells. This resulted in the replication and packaging of transcripts from the pEDI clone, derived from transfected cDNA.
  • Sequencing of the pEDI clone revealed that the DI RNA comprised of three sections from the EAV genome which were fused together in frame relative to the replicase reading frame.
  • Interestingly, the DI RNA retained all sequences encoding the structural proteins found in the EAV.

Analysing the Sequences Required for Replication and Packaging

  • The researchers drilled down to pinpoint the sequences required for replication/DI-based transcription and the packaging of EAV DI RNAs. This process was aided by inserting a reporter gene, the chloramphenicol acetyltransferase, into the DI RNA genome.
  • The study thereby achieved in reducing the maximum size of a replication-competent EAV DI RNA genome to approximately 3 kb, enhancing our understanding of the replication and packaging process of the EAV DI RNAs.

Cite This Article

APA
Molenkamp R, Rozier BC, Greve S, Spaan WJ, Snijder EJ. (2000). Isolation and characterization of an arterivirus defective interfering RNA genome. J Virol, 74(7), 3156-3165. https://doi.org/10.1128/jvi.74.7.3156-3165.2000

Publication

Journal of virology
ISSN: 0022-538X
NlmUniqueID: 0113724
Country: United States
Language: English
Volume: 74
Issue: 7
Pages: 3156-3165

Researcher Affiliations

Molenkamp, R
  • Department of Virology, Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
Rozier, B C
    Greve, S
      Spaan, W J
        Snijder, E J

          MeSH Terms

          • Animals
          • Cell Line
          • Chloramphenicol O-Acetyltransferase / genetics
          • Cloning, Molecular
          • Cricetinae
          • DNA, Complementary
          • Equartevirus / genetics
          • Equartevirus / physiology
          • Genome, Viral
          • Helper Viruses / genetics
          • Helper Viruses / physiology
          • RNA, Viral / genetics
          • Serial Passage
          • Virus Replication / genetics

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