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Journal of Virology, June 2003, p. 6720-6730, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6720-6730.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Stem-Loop III in the 5' Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication

Sharmila Raman, Peter Bouma,{dagger} Gwyn D. Williams, and David A. Brian*

Department of Microbiology, University of Tennessee, College of Veterinary Medicine, Knoxville, Tennessee 37996-0845

Received 13 August 2002/ Accepted 24 March 2003

Higher-order structures in the 5' untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5' UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of -9.1 kcal/mol in the positive strand and -3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5' UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that its associated intra-5' UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.

* Corresponding author. Mailing address: Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845. Phone: (865) 974-4030. Fax: (865) 974-4007. E-mail: dbrian{at}utk.edu.

{dagger} Present address: Preventive Medicine and Biometrics Department, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

Journal of Virology, June 2003, p. 6720-6730, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6720-6730.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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