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Journal of Virology, January 2001, p. 125-133, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.125-133.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

cis-Acting Sequences Required for Coronavirus Infectious Bronchitis Virus Defective-RNA Replication and Packaging

Kevin Dalton,1,dagger Rosa Casais,1 Kathy Shaw,1 Kathleen Stirrups,1,Dagger Sharon Evans,1 Paul Britton,1 T. David K. Brown,2 and Dave Cavanagh1,*

Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN,1 and Department of Pathology, Division of Virology, University of Cambridge, Cambridge CB2 1QP,2 United Kingdom

Received 26 June 2000/Accepted 6 October 2000

The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

* Corresponding author. Mailing address: Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, United Kingdom. Phone: 44 1635 577273. Fax: 44 1635 577263. E-mail: dave.cavanagh{at}bbsrc.ac.uk.

dagger Present address: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.

Dagger Present address: University of Cambridge, Department of Haematology, Division of Transfusion Medicine, Cambridge CB2 2PT, United Kingdom.

Journal of Virology, January 2001, p. 125-133, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.125-133.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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