Related Experiment Videos
An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA
L C van Dinten1, J A den Boon, A L Wassenaar
1Department of Virology, Institute of Medical Microbiology, Leiden University, The Netherlands.
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.
Insights
Researchers created an infectious full-length equine arteritis virus (EAV) cDNA clone. This tool enables foreign gene expression and reveals distinct requirements for viral replication and mRNA synthesis.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Equine arteritis virus (EAV) is a positive-strand RNA virus.
- EAV employs a discontinuous transcription mechanism for expressing structural genes via subgenomic mRNAs.
- Understanding EAV's replication and transcription is crucial for viral control.
Purpose of the Study:
- To construct a full-length infectious cDNA clone of the EAV genome.
- To investigate the potential of the cDNA clone for foreign gene expression.
- To explore the relationship between EAV genome replication and subgenomic mRNA synthesis.
Main Methods:
- Construction of a stable bacterial plasmid (pEAV030) containing the full-length EAV genome cDNA.
- In vitro transcription and electroporation of RNA into BHK-21 cells to assess infectivity.
- Introduction and recovery of genetic marker mutations.
- Expression of the chloramphenicol acetyltransferase (CAT) reporter gene.
Main Results:
- Infectious EAV RNA was generated from the corrected cDNA clone (pEAV030).
- A genetic marker was successfully introduced and recovered, validating the system.
- The EAV clone demonstrated efficient foreign gene expression (CAT gene).
- A specific point mutation allowed RNA replication but blocked subgenomic mRNA production.
Conclusions:
- The pEAV030 clone serves as a valuable tool for EAV reverse genetics and foreign gene expression.
- The study provides the first experimental evidence for separable requirements for arterivirus genome replication and discontinuous mRNA synthesis.