The intraleader AUG nucleotide sequence context is important for equine arteritis virus replication.

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• The 5' leader sequence of equine arteritis virus (EAV) contains an intraleader open reading frame (ILO).
• The ILO start codon is in a suboptimal context for protein synthesis initiation.

Purpose of the Study:

• To investigate the significance of the EAV ILO and its encoded leader peptide.
• To determine the role of ILO translation in the EAV replication cycle.

Main Methods:

• Generated expression plasmids and transcribed capped RNA for in vitro translation.
• Utilized site-directed mutagenesis to alter the ILO start codon context.
• Created infectious EAV cDNA clones with modified ILO start codons.
• Assessed viral replication kinetics, viral yields, and plaque sizes in BHK-21 cells.
• Analyzed viral sequence and phenotype after passaging.

Main Results:

• In vitro translation confirmed leader peptide synthesis and its inverse effect on downstream N protein translation.
• Mutant EAV viruses with optimized or incapacitated ILO start codons were viable but showed reduced replication kinetics and viral yields.
• Mutations were rapidly repaired upon passaging, restoring the wild-type sequence and phenotype.
• The leader peptide itself was dispensable for EAV replication.

Conclusions:

• EAV replication is independent of intraleader peptide synthesis.
• The ILO 5' nucleotide sequence, rather than its translation, is crucial for EAV replication.
• The leader peptide appears to have no discernible function in the EAV life cycle.