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Proceedings of the National Academy of Sciences of the United States of America1997; 94(3); 991-996; doi: 10.1073/pnas.94.3.991

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription.

Abstract: Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.
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Publication Date: 1997-02-04 PubMed ID: 9023370PubMed Central: PMC19627DOI: 10.1073/pnas.94.3.991Google 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 article presents findings from an experiment exploring the role of arterivirus—the virus responsible for equine arteritis—in RNA genome replication and mRNA synthesis, demonstrating a certain point mutation prevents the virus from producing the necessary subgenomic mRNAs for protein construction.

Research Background

  • The Equine arteritis virus (EAV) is a unique type of RNA virus that uses a specialized transcription method to create a set of six subgenomic mRNA (messenger RNA), essential for expressing structural genes.

Research Methodology

  • The researchers created a stable bacterial plasmid named pEAV030, which contains a full-length cDNA (complementary DNA) copy of the EAV genome.
  • A single point mutation in the replicase gene of the genome was removed. The replicase gene is responsible for dictating viral replication.
  • RNA transcripts created from pEAV030 were found to be infectious after being transferred to BHK-21 cells using electroporation—a technique used to introduce foreign genetic material into cells.
  • A genetic marker mutation was introduced at the cDNA level and later recovered from the progeny virus, demonstrating the mutation process.
  • The potential of pEAV030 as a tool to express foreign genes was displayed through the effective expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs.

Research Findings

  • The original point mutation that rendered the full-length clone non-infectious showed an interesting outcome: While RNA with this mutation can replicate efficiently, it does not produce the necessary subgenomic mRNAs for structural protein expression. In other words, the mutation disrupted the virus’ ability to produce proteins.
  • This finding provided the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis can partially differ, and these processes can be separated in an experimental setting.

Implications of the Research

  • This research deepens scientific understanding of the Equine arteritis virus, particularly how it transcribes RNA and mRNA and how a point mutation can disrupt protein synthesis processes.
  • The discovery that replicase gene mutation prevents mRNA production while allowing genome replication suggests a possible intervention point in mitigating the impacts of diseases related to arterivirus.
  • The study also demonstrates the potential use of plasmid pEAV030 in expressing foreign genes—broadening the scope for further research in genetic manipulation and gene therapy.

Cite This Article

APA
van Dinten LC, den Boon JA, Wassenaar AL, Spaan WJ, Snijder EJ. (1997). An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci U S A, 94(3), 991-996. https://doi.org/10.1073/pnas.94.3.991

Publication

Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
NlmUniqueID: 7505876
Country: United States
Language: English
Volume: 94
Issue: 3
Pages: 991-996

Researcher Affiliations

van Dinten, L C
  • Department of Virology, Institute of Medical Microbiology, Leiden University, The Netherlands.
den Boon, J A
    Wassenaar, A L
      Spaan, W J
        Snijder, E J

          MeSH Terms

          • Animals
          • Cell Line
          • Cloning, Molecular
          • Cricetinae
          • DNA, Complementary / genetics
          • Equartevirus / enzymology
          • Equartevirus / genetics
          • Gene Expression Regulation, Viral / genetics
          • Genetic Vectors
          • Kidney
          • Molecular Sequence Data
          • Point Mutation
          • RNA, Messenger / biosynthesis
          • RNA, Messenger / genetics
          • RNA, Viral / biosynthesis
          • RNA, Viral / genetics
          • RNA-Dependent RNA Polymerase / genetics
          • Transcription, Genetic / genetics
          • Virus Replication

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