Stem-loops III and IV in the 5' untranslated region are CIS-acting elements in bovine coronavirus DI RNA replication

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, and transcription. The bovine coronavirus (BCo V) 210 nucleotide (nt) 5' UTR is predicted to contain four higher-order structures identified as stem-loops I to IV. In this study, evidence supporting the structure and a cis-acting function in BCoV defective interfering (DI) RNA replication of two of these structures, stem-loops III and IV is presented. Both stem-loops show phylogenetic conservation among group 2 coronaviruses and appear to have homologs or analogs in coronavirus groups 1 and 3 and in the SARS coronavirus. Stem-loop III has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5' UTR open reading frame (ORF). Stemloop III in BCoV DI RNA (i) maps at nt 97 through 116, and has associated with it beginning at nt 100 an ORF potentially encoding an 8 amino-acid peptide, (ii) must maintain stem integrity in the positive and in the negative strands for DI RNA replication, (iii) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation, (iv) specifically binds in the positive strand the 50 kDa viral nucleocapsid protein, and an unidentified viral protein of approximately 22 k.Da, and (v) in the negative strand specifically binds seven cellular proteins in the range of 76 to 25 kDa. These results indicate that stem-loop III might function as a cis-acting element for DI RNA replication through interactions in the positive strand with viral proteins and in the negative strand with cellular proteins. Stem-loop IV in BCo V DI RNA (i) maps between nts 186 and 215, (ii) must maintain stem integrity only in the positive strand for DI RNA replication, and (iii) specifically binds eight cellular proteins in the range of 75 to 25 kDa. These results indicate that stem-loop IV might function as a cisacting element for DI RNA replication through interactions with cellular proteins. It is postulated that stem-loops III and IV function similarly in the virus genome.