Effect of intergenic consensus sequence flanking sequences on coronavirus transcription

Area of Science:

• Virology
• Molecular Biology
• RNA Transcription

Background:

• Mouse hepatitis virus (MHV) defective interfering (DI) RNAs are transcribed into subgenomic RNAs in helper virus-infected cells.
• Previous studies showed that inserting a specific intergenic sequence into MHV DI RNA leads to subgenomic DI RNA transcription.

Purpose of the Study:

• To investigate the role of flanking sequences in MHV RNA transcription.
• To identify the minimum intergenic sequence required for MHV transcription.

Main Methods:

• Construction of DI cDNAs with varying lengths of upstream and downstream flanking sequences around the 18-nucleotide intergenic region.
• Analysis of intracellular genomic and subgenomic DI RNA species ratios.
• Construction of DI cDNAs with deletions within the intergenic sequence to determine the minimum functional sequence.

Main Results:

• Flanking sequences (0 to 1,440 nucleotides upstream, 0 to 1,671 nucleotides downstream) did not significantly affect the ratios of subgenomic to genomic DI RNA.
• The UCUAAAC and GCUAAAC sequences were sufficient for small amounts of subgenomic DI RNA synthesis.
• No subgenomic DI RNA transcription was observed with UCUAAAG and GCTAAAG sequences.

Conclusions:

• Sequences flanking the intergenic region between MHV genes 6 and 7 do not regulate subgenomic DI RNA transcription.
• The UCUAAAC consensus sequence is sufficient for subgenomic DI RNA transcription.