Structural basis for cloverleaf RNA-initiated viral genome replication.

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• Positive-strand RNA virus genomes encode both protein translation and replication machinery.
• Enteroviruses utilize a 5' cloverleaf RNA structure to regulate the switch between translation and replication.
• This switch involves interactions with host poly(C)-binding protein 2 (PCBP2) and viral 3CDpro protein.

Purpose of the Study:

• To determine the structures of enterovirus cloverleaf RNA from coxsackievirus and poliovirus.
• To elucidate the mechanism by which cloverleaf RNA acts as a molecular switch for viral replication.
• To investigate the role of the A•C-U base triple and PCBP2 binding sites in regulating this switch.

Main Methods:

• X-ray crystallography to determine cloverleaf RNA structures.
• Biophysical assays to assess protein-RNA interactions and binding affinities.
• Structural analysis of RNA folding, base pairing, and protein binding interfaces.

Main Results:

• Cloverleaf RNA folds into an H-type four-way junction stabilized by a unique A•C-U base triple.
• PCBP2 binding sites are spatially proximal and distinct from the 3CDpro binding site.
• The A•C-U base triple restricts cloverleaf flexibility, partially occluding PCBP2 binding; its elimination enhances PCBP2 affinity.

Conclusions:

• A novel mechanistic model for the enterovirus cloverleaf RNA acting as a molecular switch is proposed.
• The A•C-U base triple plays a critical role in regulating PCBP2 binding and the translation-replication switch.
• Structural insights provide a foundation for understanding viral replication control in enteroviruses.