A novel point mutation (L70P) inactivates poliovirus 3C protease

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Poliovirus (PV) RNA genome is translated into a polyprotein processed by viral proteases.
• The major viral protease, 3C, is essential for processing viral proteins and viral replication.
• Understanding 3C protease function is critical for PV research and antiviral development.

Purpose of the Study:

• To investigate the functional impact of a novel 3C protease mutation (L70P) identified during PV protein expression.
• To determine the effect of the L70P mutation on PV replication and viral particle formation.
• To elucidate the structural basis for the loss of 3C protease activity.

Main Methods:

• Expression of PV 3ABC protein in Escherichia coli to identify mutants.
• Generation of recombinant adenoviruses expressing wild-type and mutant 3C protease.
• Recovery of infectious PV from full-length PV cDNA containing the L70P mutation.
• Complementation studies using PV cDNA with a 1AB deletion.
• Structural analysis of the mutant 3C protein.

Main Results:

• The L70P mutation was identified in PV 3C protease, resulting in a complete loss of protease activity.
• Infectious PV could not be recovered from PV cDNA harboring the L70P mutation.
• The L70P mutant 3C protease retained the ability to complement PV cDNA with a 1AB deletion, indicating partial function.
• Structural analysis revealed that the L70P mutation disrupts a key hydrogen bond near the catalytic site, potentially causing strain.

Conclusions:

• The L70P mutation inactivates poliovirus 3C protease due to its proximity to the catalytic site.
• This mutation provides insights into the structure-function relationship of 3C protease.
• The findings contribute to understanding PV replication mechanisms and potential antiviral targets.