The Intraleader AUG Nucleotide Sequence Context is Important for Equine Arteritis Virus Replication

Abstract

The 5(-terminal leader sequence of the equine arteritis virus (EAV) genome contains an open reading frame (ORF) with an AUG codon in a suboptimal context for initiation of protein synthesis. To investigate the significance of this intraleader ORF (ILO), an expression plasmid was generated carrying a DNA copy of the subgenomic mRNA7 behind a T7 promoter. Capped RNA transcribed from this construct was shown to direct, in an in vitro translation system, the synthesis of leader peptide as well as N protein. Site-directed mutations aimed to either optimize or weaken the sequence context of the ILO start codon affected leader peptide synthesis as predicted; no peptide was detected when the initiation codon was incapacitated. Translation of the downstream N gene was inversely affected by leader peptide production, consistent with a ribosomal leaky scanning mechanism. To investigate the role of the leader peptide in the EAV replication life cycle we generated, using an infectious EAV cDNA clone, two mutant viruses in one of which the ILO start codon was in an optimal Kozak context for translation initiation while in the other the codon was again incapacitated. Surprisingly, both mutant viruses were equally viable and exhibited similar phenotypes in BHK-21 cells. However, their replication kinetics and viral yields were reduced relative to that of the wild-type parental virus, as were their plaque sizes. Importantly, the mutations introduced into the viruses appeared to be rapidly and precisely repaired upon passaging. Already after one viral passage a significant fraction of the viruses had regained the wild-type sequence as well as its phenotype. The results demonstrate that EAV replication is not dependent on the synthesis of the intraleader peptide. Rather, the leader peptide does not seem to have any function in the EAV life cycle. As we discuss, the available data indicate that the ILO 5( nucleotide sequence per se, not its functioning in translation initiation, is of critical importance for EAV replication.

Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5′-Untranslated-Region Mutants

“…It might be expected that in these cases, the uORF in the genome would be a regulator of not only translation but also virus replication and perhaps also virus-induced pathogenesis. A single AUG-initiated uORF is found in the genomes of arteriviruses (13,14,16) and most coronaviruses (17; this study), two families of animal positive-strand RNA viruses in the order Nidovirales (18). The role of the uORF in these viruses has undergone limited study.…”

“…In other cases, 5=-cap-dependent translation of the uORF enhances translation of the main ORF by various mechanisms (1,2,4,(7)(8)(9)(10)(11). Some plant (12) and animal (13)(14)(15) viruses that have a positive-strand (mRNA-like) genome which undergoes necessary 5=-cap-dependent translation prior to viral genome replication in the cytoplasm also have a (usually single) short uORF. It might be expected that in these cases, the uORF in the genome would be a regulator of not only translation but also virus replication and perhaps also virus-induced pathogenesis.…”

“…The role that the uORF plays in nidoviruses has been examined most closely in arteriviruses (13,14). When the AUG start codon for the uORF in equine arteritis virus, which is in a suboptimal Kozak context for translation, was changed to an AGG nonstart codon by mutation in a reverse-genetics analysis, or when the Kozak context was made optimal, the resulting virus plaque size was smaller than that of the wild type (WT), and growth kinetics were found to be impaired (13). In this case, reselection of a uORF start codon in its original suboptimal context was found upon virus passaging in cell culture.…”

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

“…Looking to the Arteriviridae family, EAV has a highly conserved AUG within the leader (present in 94/95 available genome sequences), at genomic coordinates 14-16, predicted to permit expression of a 37-amino-acid leader peptide 118 . In vitro translation of the N transcript demonstrated that the uORF is translated, and found it down-regulated translation of the downstream N ORF 119 . Mutations which disrupted this AUG or altered its predicted initiation efficiency were detrimental to viral fitness and led to rapid reversion to the WT sequence 119 , although the uORF was not essential for virus viability 119,120 .…”

In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

“…In horses the Kozak sequence is commonly found as an initiation signal for gene translation as in other vertebrates [ 21 ]. For equine arteritis virus suboptimal intraleader AUG and not an optimal Kozak sequence has been shown to be critical for virus replication [ 26 ].…”