Evidence for coronavirus discontinuous transcription

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Coronavirus subgenomic messenger RNAs (mRNAs) feature a 5'-end leader sequence originating from the genomic RNA.
• The mechanism of coronavirus transcription, specifically the role of leader sequences, remains an area of active investigation.
• Defective interfering (DI) RNAs are crucial tools for studying viral replication and transcription processes.

Purpose of the Study:

• To investigate whether coronavirus transcription involves a discontinuous transcription step.
• To determine if mouse hepatitis virus (MHV) genomic RNA leader sequences can be utilized for MHV subgenomic DI RNA transcription.
• To elucidate the trans-acting properties of genomic leader sequences in coronavirus transcription.

Main Methods:

• Construction of a novel MHV DI RNA (DI(J3-9)) with a specific leader and intergenic region.
• Infection of cells with distinct MHV helper virus variants (JHM(2) and JHM(3-9)) and MHV A59 strain.
• Sequence analysis of helper virus genomic RNAs and DI RNAs in infected cells.
• Assessment of DI RNA replication and subgenomic DI RNA synthesis.

Main Results:

• Subgenomic DI RNA synthesis was observed only in cells infected with MHV JHM(2), indicating dependence on the helper virus.
• Sequence analyses confirmed that genomic leader sequences from JHM(2) functioned in subgenomic DI RNA transcription.
• The leader sequence of subgenomic DI RNA was derived from the helper virus, demonstrating its trans-acting property.

Conclusions:

• Coronavirus transcription involves a discontinuous step, with genomic leader sequences playing a critical role.
• The genomic leader sequence exhibits a trans-acting property, essential for the efficient transcription of subgenomic DI RNAs.
• These findings provide significant insights into the molecular mechanisms of coronavirus RNA synthesis and replication.