Stem-loop III in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication

Abstract

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.

FIG. 1.

Phylogenetic and enzyme structure-probing evidence for stem-loop III. (A) Predicted secondary structures within the first 126 nt of the 210-nt 5′ UTR of the BCoV genome and the site from which 5′-end-labeled primer was extended for stem-loop III structure determination. The 65-nt BCoV leader sequence is shaded, and the leader-associated UCUAAAC intergenic sequence (nt 64 to 70) found within loop II is identified in bold. The start and stop codons for the intra-5′ UTR ORF and the start codon for ORF 1a are boxed. Previously described stem-loops I and II are identified. Stem-loop III is shown along with the variant bases found among the eight sequenced members of group 2 coronaviruses. Bases named with no superscript are found in all MHV strains (A59, 2, and JHM). Superscripts: 2, MHV-2; a, MHV-A59; j, MHV-JHM; he, HECoV-4408; e, ECoV. Stem-loop III is identical in BCoV, HEV, and HCoV-OC43. (B) Enzyme probing data for stem-loop III. Depicted is a DNA sequencing gel showing separation of primer extension products. RNase digestion conditions for the single-strand-digesting RNase T₂ and double-strand-digesting RNase CV1 are described in the text. Lanes 5 to 8 show the products of an RNA sequencing reaction done on undigested RNA to identify the base positions. (C) Summary of enzyme probing data shown in panel B.

FIG. 2.

Integrity of stem III is required for DI RNA replication as determined by analysis of mutant 1 variants of pDrep1. The mfold-predicted structures of wt and mutant transcripts of pDrep1 (pSLIII-mut1L, pSLIII-mut1R, and pSLIII-mut1L/R) in the positive and negative strands and their calculated free energies in kilocalories per mole are shown. The start codon for the intra-5′ UTR ORF is boxed (note that this moves 1 base position upstream in pSLIII-mut1L and pSLIII-mut1L/R relative to that of wt). Mutated bases are shown as lowercase letters. For Northern analyses, T7 RNA polymerase-generated transcripts of linearized plasmids were transfected into BCoV-infected cells, and RNA was extracted at the indicated times posttransfection and probed with a reporter-specific radiolabeled oligonucleotide. Accumulation of mutant molecules was determined by quantitative blotting of RNA harvested at the times indicated and at 48 h postinfection with VP1. Accumulation of pDrep1 progeny was considered to be 100%. The sequence of the RNA in the progeny replicons was determined by either bulk RT-PCR sequencing by using pDrep1-specific primers as described in the text or by first cloning and sequencing plasmid DNA from isolated colonies. Lanes labels: Uninf., mock-infected, nontransfected cells; Inf., infected, nontransfected cells; RNA, sample of the transcript. Superscript 1 indicates the sequence MACGRRFFIVVSIFISAVNSFQPGTCCILGSGPPIGHNVEDQQIRSRTTLGSRISMDV. Superscript 2 indicates that the sequence is identical to the amino acid sequence predicted for mutant pSLIII-L except that LG replace RR at positions 5 and 6.

FIG. 3.

Integrity of stem III is required for DI RNA replication as determined by analysis of mutant 2 variants of pDrep1. The assays depicted were carried out as described in the legend to Fig. 2, except that mutant variants (pSLIII-mut2L, pSLIII-mut2R, and pSLIII-2L/R) were used.

FIG. 4.

Importance of stem III for DI RNA replication independent of the potential short ORF product. The importance of stem III in the presence of little (A and B) or no (C) change in the potential short ORF product was measured by assays described in the legend to Fig. 2. (A) Accumulation and sequence of progeny from wt or mutant transcripts bearing a mutation at base 109 (pSLIII-mutG109→A). (B) Accumulation and sequence of progeny from wt or mutant transcripts bearing a mutation at base 110 (pSLII0-mutG110→A). (C) Accumulation and sequence of progeny of wt or mutants with changes at codon wobble positions that maintain the wt ORF product MLVGVDFS. Stem III was maintained in pSLIII-mut5 but was disrupted in transcripts of pSLIII-mut6. Assays were carried out as described in the legend to Fig. 2.

FIG. 5.

Stem-loop III-associated short ORF correlates with maximal DI RNA accumulation. (A) Accumulation and sequence of progeny from DI RNA in which the stem-loop III-associated short ORF begins with an AcG threonine codon (pSLIII-mut3L). The sequence of the progeny in VP1 was determined to be a mixture by sequencing the asymmetrically amplified RT-PCR product. (B) Accumulation and sequence of mutant transcripts in which the stem-loop III-associated short ORF begins with a uaG stop codon (pSLIII-4L/R). The sequence of progeny molecules in VP1 was determined from clones of the RT-PCR product. Assays were carried out as described in the legend to Fig. 2.

FIG. 6.

Potential stem-loop III homologs in coronavirus groups 1 and 3 are predicted by the Zuker mfold algorithm. AUG codons for potential stem-loop-associated ORFs are boxed. In HCoV-229E the second AUG is in the +1 frame relative to the first ORF.