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A cis-acting function for the coronavirus leader in defective interfering RNA replication
R Y Chang1, M A Hofmann, P B Sethna
1Department of Microbiology, University of Tennessee, Knoxville 37996-0845.
Journal of Virology
|December 1, 1994
Summary
The coronavirus leader RNA sequence alone is insufficient for replication. However, a minimal structure within the leader is essential for defective interfering RNA replication, with a preference for wild-type sequences.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Coronaviruses utilize subgenomic mRNAs for replication, with the 5' leader sequence hypothesized as a cis-acting replication signal.
- Understanding coronavirus RNA replication mechanisms is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the role of the 5' leader sequence in bovine coronavirus RNA replication.
- To determine if the leader sequence alone is sufficient for replication or if additional elements are required.
Main Methods:
- Transfection of synthetic RNA transcripts (N mRNA and defective interfering RNA) into helper virus-infected cells.
- Analysis of RNA replication and packaging using reporter gene expression and molecular assays.
- Site-directed mutagenesis to assess the importance of the leader's secondary structure (stem-loop).
Main Results:
- The 65-nucleotide leader sequence alone was insufficient for the replication of synthetic N mRNA transcripts.
- Synthetic defective interfering (DI) RNA transcripts replicated and were packaged, indicating the leader is necessary but not solely sufficient.
- Disruption of the intraleader stem-loop abolished replication, but base substitutions minimally impaired it, suggesting a preference for wild-type leader sequences after initial replication.
- Bovine coronavirus DI RNA did not require downstream sequence elements for replication or packaging, unlike some other coronaviruses.
Conclusions:
- The 5' leader sequence is a necessary component of the cis-acting replication signal for bovine coronavirus DI RNA.
- A minimal structural element within the leader is required for replication initiation.
- The wild-type leader sequence is strongly preferred during subsequent replication rounds, indicating a potential mechanism for maintaining genomic integrity.
- Bovine coronavirus DI RNA replication and packaging are independent of downstream genomic sequences, differentiating it from other coronavirus systems.