Unusual loop-sequence flexibility of the proximal RNA replication element in EMCV

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Picornaviruses possess stable RNA structures (OriL and OriR) essential for replication.
• The OriL element in enteroviruses forms a cloverleaf structure, critical for poliovirus replication.
• Limited information exists regarding cardiovirus OriL structures.

Purpose of the Study:

• Investigate the structural features of the stem-loop A (SL-A) apical loop in mengovirus (a cardiovirus).
• Determine the sequence requirements of the SL-A apical loop for mengovirus RNA replication.
• Elucidate the role of SL-A structure in viral replication and reproduction.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to characterize the mengovirus SL-A apical loop structure.
• In vivo SELEX (Systematic Evolution of Ligands by Exponential Enrichment) experiments to assess sequence tolerance and functional requirements.
• Mutagenesis studies varying SL-A loop size and stem length to evaluate impact on viral replication.

Main Results:

• The mengovirus SL-A apical loop was identified as an octaloop using NMR.
• In vivo SELEX revealed significant sequence tolerance within the octaloop for supporting virus replication.
• Mutational analysis demonstrated that both stem length and octaloop stability are critical for viral RNA replication and virus reproduction.

Conclusions:

• Stem-loop A is a vital component for mengovirus replication.
• The octaloop's sequence flexibility suggests it does not mediate sequence-specific RNA-protein or RNA-RNA interactions.
• Stem length and octaloop stability are key determinants for efficient viral RNA replication and virus reproduction.