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

Abstract

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.