Studies of murine coronavirus DI RNA replication from negative-strand transcripts

Area of Science:

• Virology
• Molecular Biology
• RNA Replication

Background:

• Mouse hepatitis virus (MHV) defective interfering (DI) RNAs are known to replicate and accumulate in infected cells.
• The mechanism of DI RNA replication and the factors influencing its characteristics are not fully understood.

Purpose of the Study:

• To investigate the role of transcript polarity and 3' end modifications in the replication and leader sequence switching of MHV DI RNAs.
• To elucidate the specific stage of RNA synthesis where leader switching occurs.

Main Methods:

• Introduction of positive- and negative-strand MHV DI RNA transcripts into MHV-infected cells.
• Analysis of accumulated DI RNA replication products.
• Sequencing of leader regions of replicated DI RNAs to identify "leader switching" events.

Main Results:

• Both positive- and negative-strand MHV DI RNA transcripts supported DI RNA replication and accumulation.
• Leader sequence switching to the helper virus sequence was observed in the majority of DI RNAs derived from negative-strand transcripts lacking extra 3' nucleotides.
• Leader sequence switching was significantly reduced in DI RNAs derived from negative-strand transcripts with an additional 4 non-MHV nucleotides at the 3' end.
• Leader switching was less frequent in positive-strand DI RNAs compared to negative-strand DI RNAs.

Conclusions:

• The presence of extra nucleotides at the 3' end of negative-strand DI RNA transcripts inhibits leader sequence switching.
• Leader sequence switching on MHV DI RNA likely occurs during the synthesis of positive-strand RNA molecules.