Higher-order RNA structures in the 5= untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ϳ36% nucleotide sequence divergence. In a previous study, each of three 5=-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stemloops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ϳ200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5= UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5= UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5= UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture. In positive-strand RNA viruses that replicate in the cytoplasm, genomic 5=-end-proximal RNA structures carry out several functions required for virus reproduction. In coronaviruses, these are thought to include (i) translation initiation, commonly presumed to occur by a canonical cap-dependent 5=-terminal ribosomal entry mechanism, to synthesize the replicase enzymes from open reading frame 1 (13, 14, 31); (ii) signaling an RNA-dependent RNA polymerase template switch during minus-strand synthesis at a heptameric transcription-regulatory sequence (UCUAAAC in the case of mouse hepatitis coronavirus [MHV] and bovine coronavirus [BCoV]) (Fig. 1A and B) for placement of a common leader on subgenomic mRNAs (sgmRNAs) (43,48,49,55); (iii) encoding signals on the 3= end of minus-strand genomic RNA (the antigenome) for initiating synthesis of plus-strand genomic mRNAs and sgmRNAs (9, 43, 48, 55); (iv) possibly harboring signals that act in trans to initiate synthesis of nascent plus-strand genomes (46); (v) possibly directly influencing initiation of minus-strand synthesis at the 3= end of the genome (33); and (vi) harboring a genome packaging signal (10,18). A mechanistic understanding of these events may aid in the devel...

An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5= untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5=-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5=-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.

Genomes of positive (؉)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5=-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5= untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5= untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (؉) strand. We postulate that the higher-order structure promotes (؉)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH 2 -proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate. What constitutes the cis-acting requirements for coronavirus RNA replication has remained an intriguing question since it was discovered that the subgenomic mRNAs (sgmRNAs) of coronaviruses (used primarily to synthesize viral structural proteins) are both (i) 5= and 3= coterminal with the genome for at least ϳ70 and 1,670 nucleotides (nt), respectively, lengths greater than those of many viral RNA polymerase promoters (1-3), and (ii) are present in sgmRNA-length replication-intermediate-like doublestranded RNA structures that are involved in sgmRNA synthesis (4-6) yet fail to replicate when transfected, as synthetic transcripts, into virus-infected cells (Fig. 1) (7). If replication of the coronavirus sgmRNAs normally occurs during infection, it might be expected that they would replicate following their transfection into virus-infected cells, since all trans-acting factors required for viral RNA replication are present. In coronaviruses, the 5= twothirds of the single-stranded positive (ϩ)-strand ϳ30-kb coronavirus genome is used as mRNA for synthesis of overlapping polyproteins 1a (ϳ4,000 amino acids [aa]) and 1ab (ϳ7,000 aa), which are proteolytically processed into the 16 replicase proteins that make up the replication/transcription complex, whereas the 3= one-third of the genome is transcribed into a 3= nested set of sgmRNAs that are coterminal with ...