Sequence and secondary structure requirements in a highly conserved element for foot-and-mouth disease virus internal...

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Picornaviruses, including Foot-and-mouth disease virus (FMDV), utilize an internal ribosome entry site (IRES) for cap-independent translation initiation.
• Eukaryotic translation initiation factors (eIFs), specifically eIF4G and eIF4B, are essential for mediating ribosome recruitment to the viral RNA IRES.

Purpose of the Study:

• To investigate the primary sequence and secondary structure requirements of a conserved element within the FMDV IRES for efficient IRES function.
• To understand the role of this conserved element in the binding of eIF4G and subsequent IRES-directed translation.

Main Methods:

• Site-directed mutagenesis was employed to alter the conserved sequence element within the FMDV IRES.
• Assays were performed to assess eIF4G binding to the mutated IRES elements.
• FMDV IRES-driven translation activity was measured in vitro.

Main Results:

• Mutations within two unpaired dinucleotide stretches significantly impaired both eIF4G binding and IRES-directed translation.
• Maintenance of double-stranded secondary structure in base-paired regions was critical for IRES function.
• In some cases, the specific nucleotide sequence within these double-stranded regions also influenced IRES activity.
• Supplementation with additional eIF4F complex could not rescue the impaired IRES activity or eIF4G binding.

Conclusions:

• The conserved sequence element in the FMDV IRES possesses critical structural and sequence features essential for eIF4G binding and translation initiation.
• Disruptions to the secondary structure of this element cannot be compensated by increased initiation factor concentrations, highlighting its specific structural role.
• These findings provide insights into the intricate mechanisms of picornavirus translation and potential targets for antiviral strategies.