Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• RNA viruses rely on RNA-dependent RNA polymerase (RdRp) for replication.
• RdRp fidelity, the accuracy of nucleotide incorporation, is critical for viral genome stability and can be a target for antiviral strategies.
• Perturbing RdRp fidelity is a potential avenue for rational vaccine design.

Purpose of the Study:

• To investigate the molecular mechanisms underlying nucleotide incorporation fidelity in poliovirus RdRp.
• To compare the conformational dynamics of wild-type (WT) RdRp with a high-fidelity mutant (H273R).
• To elucidate how enzyme dynamics relate to the fidelity checkpoint during nucleotide selection.

Main Methods:

• X-ray crystallography to determine enzyme structures.
• Molecular dynamics simulations to model enzyme behavior.
• NMR spectroscopy to probe enzyme dynamics and nucleotide interactions.
• Pre-steady-state kinetics to measure enzyme reaction rates.

Main Results:

• The nucleotide-binding site of RdRp exists in two states: occluded and competent.
• Primed template RNA binding enhances conformational dynamics between these states.
• Wild-type RdRp favors the occluded state, promoting fidelity; the H273R mutant favors the competent state, increasing errors.
• NMR data indicated Met-187 resonance reflects the enzyme's ability to verify nucleotide correctness.
• Kinetic data support the role of conformational dynamics in the fidelity checkpoint.

Conclusions:

• Faithful nucleotide incorporation is achieved by linking the equilibrium of nucleotide-binding pocket conformations and active site dynamics to the correctness of the bound nucleotide.
• Understanding these dynamics is essential for the rational design of viral vaccine candidates by manipulating RdRp fidelity.
• Multiple biophysical and biochemical approaches are necessary to fully understand polymerase fidelity mechanisms.