An in vitro system for the leader-primed transcription of coronavirus mRNAs.

AUTOR(ES)

Baker, S C

RESUMO

We have developed an in vitro transcription system which can utilize exogenous leader RNA for mouse hepatitis virus (MHV) 'leader-primed' mRNA transcription. Cytoplasmic extracts containing viral proteins and template RNA were prepared by lysolecithin permeabilization of MHV-infected cells. Synthetic leader RNA which differed in sequence from the endogenous leader RNA was added to the extracts and demonstrated to be incorporated into MHV mRNAs. Irrespective of the size of leader RNAs added, the exogenous leader RNA was joined to the endogenous mRNA at the same site, which corresponds to a UCUAA pentanucleotide repeat region. Only leader RNAs containing the pentanucleotide sequences could be utilized for transcription. Mismatches between the intergenic site and the exogenous leader sequence within the pentanucleotide repeat region were corrected in the in vitro system. This in vitro system thus established a novel mechanism of leader-primed transcription using exogenous RNA in trans, and suggests the involvement of a specific ribonuclease activity during coronavirus mRNA synthesis.

Documentos Relacionados

• Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs.
• Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription.
• Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.
• Differential effect of ATP concentration on synthesis of vesicular stomatitis virus leader RNAs and mRNAs.
• Globin mRNAs are primers for the transcription of influenza viral RNA in vitro