Characterization of a murine coronavirus defective interfering RNA internal cis-acting replication signal

Young Nam Kim, Shinji Makino

Research output: Contribution to journalArticle

43 Scopus citations


The mouse hepatitis virus (MHV) sequences required for replication of the JHM strain of MHV defective interfering (DI) RNA consist of three discontinuous genomic regions: about 0.47 kb from both terminal sequences and a 0.13-kb internal region present at about 0.9 kb from the 5' end of the DI genome. In this study, we investigated the role of the internal 0.13-kb region in MHV RNA replication. Overall sequences of the 0.13-kb regions from various MHV strains were similar to each other, with nucleotide substitutions in some strains; MHV-A59 was exceptional, with three nucleotide deletions. Computer-based secondary-structure analysis of the 0.13-kb region in the positive strand revealed that most of the MHV strains formed the same or a similar main stem-loop structure, whereas only MHV-A59 formed a smaller main stem-loop structure. The RNA secondary structures in the negative strands were much less uniform among the MHV strains. A series of DI RNAs that contained MHV-JHM-derived 5'- and 3'-terminal sequences plus internal 0.13- kb regions derived from various MHV strains were constructed. Most of these DI RNAs replicated in MHV-infected cells, except that MRP-A59, with a 0.13- kb region derived from HV-A59, failed to replicate. Interestingly, replication of MRP-A59 was temperature dependent; it occurred at 39.5°C but ont at 37 or 35°C, whereas a DI RNA with an MHV-JHM-derived 0.13-kb region replicated at all three temperatures. At 37°C, synthesis of MRP-A59 negative-strand RNA was detected in MHV-infected and MRP-A59 RNA-transfected cells. Another DI RNA with the internal 0.13-kb region deleted also synthesized negative-strand RNA in MHV-infected cells. MRP-A59-transfected cells were shifted from 39.5 to 37°C at 5.5 h postinfection, a time when most MHV negative-strand RNAs have already accumulated; after the shift, MRP- A59 positive-strand RNA synthesis ceased. The minimum sequence required for maintenance of the positive-strand major stem-loop structure and biological function of the MHV-JHM 0.13-kb region was about 57 nucleotides. Function was lost in the 50-nucleotide sequence that formed a positive-strand stem-loop structure identical to that of MHV-A59. These studies suggested that the RNA structure made by the internal sequence was important for positive-strand MHV RNA synthesis.

Original languageEnglish (US)
Pages (from-to)4963-4971
Number of pages9
JournalJournal of virology
Issue number8
StatePublished - Aug 1995

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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