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. 2014 Jan;88(2):846-58.
doi: 10.1128/JVI.02831-13. Epub 2013 Oct 30.

Reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus 5'-untranslated-region mutants

Hung-Yi Wu  1 , Bo-Jhih Guan, Yu-Pin Su, Yi-Hsin Fan, David A Brian
Affiliations

Reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus 5'-untranslated-region mutants

Hung-Yi Wu et al. J Virol. 2014 Jan.

Abstract

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.

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Figures

FIG 1
FIG 1
MHV genomic 5′ UTR. (A) MHV genome and subgenomic mRNAs. A uORF is found within the 5′ UTR of the genome but not sgmRNAs. ORF1 is translated from the genome beginning at nt 210 to produce a polyprotein that is co- and posttranslationally processed into 16 replicase-related nonstructural proteins. The 3′ nested set of sgmRNAs is translated to produce the virion structural proteins. A pseudoknot-induced −1 frameshifting event at the ORF1a/1b junction during translation maintains an optimal ratio of ORF1a and ORF1b proteins for virus replication. The filled bar at the 5′ terminus of each mRNA species represents the common leader that is encoded only at the genomic 5′ end. (B) RNA structures in the MHV genomic 5′ UTR. Shown are stem-loops 1 through 5 identified by bioinformatic, genetic, and physical structure analyses. Nucleotides 140 through 170 form a long-range RNA-RNA interaction with downstream nt 332 through 363 (not shown). The underlined heptameric sequence UCUAAAC in stem-loop 3 at the 3′ terminus of the leader is the core RdRp template-switching signal that directs leader acquisition on MHV sgmRNAs. Boxes identify the uORF start codon (nt 99), the genomic ORF1 start codon (nt 210), and a second nearby potential alternate ORF1 start codon (nt 219) as well as three in-frame stop codons for the uORF. Positions used for deleting regions of stem-loop 4 (nt 96 through 115, 91 through 120, 80 through 130, and 75 through 138) are identified. Potential CUG-initiated translation start sites in frame with the uORF and ORF1 are found beginning at nt 111 and 159.
FIG 2
FIG 2
Disruptive point mutations in the uORF and subsequent reselection of the uORF. (A) Description of mutations in M1 through M6. ORFs are identified by shading. Mutated nucleotides are identified by boldface type. Bold arrowheads identify positions of WT start codons. The naturally occurring translation start and stop codons are underlined. Nucleotides are numbered beginning with the genome 5′ end. (B) Summary of WT and mutant recombinant virus behavior for M1 through M6. VP, virus passage; NA, not applicable. (C) Electrophoresis of radiolabeled proteins from in vitro (RRL) translation reactions in one representative experiment. (Top) SDS-PAGE of in vitro-synthesized nsp1 protein or the uORF-nsp1 fusion protein from 100 ng of RNA transcript. Quantitation was determined by scintillation counting of excised bands. (Middle) Percentage of methionine-normalized counts relative to those in the WT band. (Bottom) Separate ethidium bromide-stained agarose gel showing electrophoretically separated RNA from 500 ng loaded per lane. (D) A single growth kinetics analysis where the MOI was 1.0 for the WT and M1 through M6. (E) Plaques of WT, M1, M2, M3, M4, and M6 viruses.
FIG 3
FIG 3
Deletion mutations and subsequent reselection of uORFs in progeny virus. (A) WT sequence positions of stem-loops 3 and 4 as noted in Fig. 1. The uORF is shown by shading. The heptameric RdRp template-switching signal, UCUAAAC, is underlined. In mutant virus MΔ96–115, the C80U transition causing a new uAUG in virus passage 10 is identified with a ↓. In mutant virus MΔ91–120, the A77G transition causing a new uAUG in virus passage 10 is identified with a ↓. In MΔ80–130, a 4-nt insertion, AUCU, occurs between nt 57 and 58 by virus passage 10, but no new uORF is formed by this insertion. Note that this insertion creates a new UCUAA element, a spontaneous phenomenon previously described for the MHV genome near this site. With mutant MΔ75–138, no progeny virus was recovered following recombinant RNA transfection. (B) Growth kinetics analyses where the MOI was 1.0 for the WT and mutants at virus passages 1 and 10. (C) Virus plaques at 48 hpi for WT and mutant viruses at virus passage 1. (D) Northern analysis for each replicating virus using a hybridization probe that identifies a 3′-end sequence. The same number of cells was used to prepare RNA for each lane.
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