Hydrophobic pocket targeting probes for enteroviruses

Area of Science:

• Virology
• Molecular Biology
• Biophysics

Background:

• Understanding enterovirus infection dynamics, including cellular targeting and uncoating, is crucial for combating diseases like type I diabetes.
• Existing methods for visualizing virus-host interactions often compromise viral functionality.

Purpose of the Study:

• To develop and validate a novel site-specific probe for visualizing enteroviruses.
• To track enteroviruses during early infection phases and observe virus uncoating within host cells.

Main Methods:

• Development of a Pleconaril derivative probe targeting the enterovirus hydrophobic pocket.
• Conjugation of the probe to fluorescent and gold labels for microscopy (light, electron, TEM).
• Biophysical characterization (melting temperature) and biochemical assays (STD, tr-NOESY NMR) to confirm binding.
• Cellular uptake and infection studies using labeled enterovirus 1 (EV1).

Main Results:

• The probe mildly stabilized enterovirus particles and delayed uncoating but did not inhibit infection.
• Binding to echovirus 1 (EV1) and Coxsackievirus A9 (CVA9) was confirmed; binding to Coxsackie virus B3 (CVB3) was weaker.
• Molecular modeling correlated probe fit with observed binding affinities.
• Fluorescently labeled EV1 was internalized, accumulated in endosomes, and the probe separated from capsid proteins during uncoating.

Conclusions:

• The developed probes enable visualization and tracking of enteroviruses during early infection stages.
• The probe release upon uncoating offers insights into the virus uncoating process.
• This novel approach provides new avenues for studying virus-host interactions and uncoating mechanisms.