A system for study of coronavirus mRNA synthesis

A regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion

Shinji Makino, Myungsoo Joo, Jayne K. Makino

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

A system that exploits defective interfering (DI) RNAs of mouse hepatitis virus (MHV) for deciphering the mechanisms of coronavirus mRNA transcription was developed. A complete cDNA clone of MHV DI RNA containing an inserted intergenic region, derived from the area of genomic RNA between genes 6 and 7, was constructed. After transfection of the in vitro-synthesized DI RNA into MHV-infected cells, replication of genomic DI RNA as well as transcription of the subgenomic DI RNA was observed. S1 nuclease protection experiments, sequence analysis, and Northern (RNA) blotting analysis revealed that the subgenomic DI RNA contained the leader sequence at its 5′ end and that the body of the subgenomic DI RNA started from the inserted intergenic sequence. Two subgenomic DI RNAs were synthesized after inserting two intergenic sites into the MHV DI RNA. Metabolic labeling of virus-specific protein in DI RNA replicating cells demonstrated that a protein was translated from the subgenomic DI RNA, which can therefore be considered a functional mRNA. Transfection study of gel-purified genomic DI RNA and subgenomic DI RNA revealed that the introduction of genomic DI RNA, but not subgenomic DI RNA, into MHV-infected cells was required for synthesis of the subgenomic DI RNA. A series of deletion mutations in the intergenic site demonstrated that the sequence flanking the consensus sequence of UCUAAAC affected the efficiency of subgenomic DI RNA transcription and that the consensus sequence was necessary but not sufficient for the synthesis of the subgenomic DI RNA.

Original languageEnglish (US)
Pages (from-to)6031-6041
Number of pages11
JournalJournal of Virology
Volume65
Issue number11
StatePublished - Nov 1991
Externally publishedYes

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Coronavirus
Coronavirinae
RNA
Messenger RNA
synthesis
Murine hepatitis virus
genomics
Intergenic DNA
Consensus Sequence
transcription (genetics)
consensus sequence
transfection
intergenic DNA
Transfection
RNA Sequence Analysis

ASJC Scopus subject areas

  • Immunology

Cite this

A system for study of coronavirus mRNA synthesis : A regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. / Makino, Shinji; Joo, Myungsoo; Makino, Jayne K.

In: Journal of Virology, Vol. 65, No. 11, 11.1991, p. 6031-6041.

Research output: Contribution to journalArticle

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