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Related Experiment Videos

Mutagenic analysis of the coronavirus intergenic consensus sequence.

M Joo1, S Makino

  • 1Department of Microbiology, University of Texas, Austin 78712.

Journal of Virology
|November 1, 1992
PubMed
Summary

Mouse hepatitis virus (MHV) defective interfering (DI) RNA transcription is flexible, tolerating changes in the UCUAAAC consensus sequence. Leader-body fusion occurs at specific sites, challenging existing coronavirus transcription models.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • A system for mouse hepatitis virus (MHV) defective interfering (DI) RNA transcription was previously established.
  • This system involves an intergenic region from MHV inserted into MHV DI RNA, enabling subgenomic DI RNA transcription in helper virus-infected cells.

Purpose of the Study:

  • To investigate how alterations in the MHV transcription consensus sequence (UCUAAAC) affect mRNA transcription.
  • To determine the precise leader-body fusion site on subgenomic DI RNAs.

Main Methods:

  • Construction of a DI cDNA with the UCUAAAC consensus sequence.
  • Generation of 21 mutant DI RNAs by individually altering each nucleotide of the consensus sequence.
  • Analysis of subgenomic DI RNA transcription efficiency with these mutants.

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  • Sequence analysis of subgenomic DI RNAs to identify leader-body fusion sites.

    • Main Results:

    • Most UCUAAAC consensus sequence mutants supported efficient subgenomic DI RNA transcription, indicating MHV transcription flexibility.
    • Two mutants, UGUAAAC and UCGAAAC, showed reduced transcription efficiency.
    • Sequence analysis revealed leader-body fusion occurred at either the first or second nucleotide of a specific pentanucleotide sequence.

    Conclusions:

    • MHV transcription regulation demonstrates flexibility in recognizing altered consensus sequences.
    • The observed leader-body fusion sites challenge the proposed coronavirus transcription model involving RNA polymerase proofreading for nucleotide mismatch correction.