Key Points
- To purify and characterize the enzymatic activity of poliovirus polypeptide 3CD, the precursor to both the 3C proteinase and 3D RNA polymerase.
- Cloned poliovirus type 2 (Sabin) cDNA encoding 3CD into a bacterial T7 expression system.
- Engineered a site-specific Thr-to-Lys mutation at the 3C/3D cleavage site (3CDpro T181K) to minimize autoprocessing and purified the resulting mutant polypeptide to 86% homogeneity.
- Evaluated the proteolytic capability of purified 3CD against capsid precursor P1, nonstructural precursor 2BC, and synthetic peptides, alongside testing for RNA polymerase activity.
- Purified mutant 3CDpro cleaves structural P1 capsid precursors, nonstructural 2BC precursor polypeptides, and synthetic 2BC cleavage site peptides.
- The detergent sensitivity of P1 processing mediated by purified mutant 3CD matches that observed in poliovirus-infected HeLa cell lysates.
- Purified 3CD displays no detectable RNA polymerase activity, whereas 3Dpol separated during purification exhibits active polymerase function.
Structured PICO
PPopulationPoliovirus type 2 (Sabin) 3CD proteinase expressed in bacteria using a T7 expression system
IInterventionSite-specific mutagenesis of the 3C/3D cleavage site (specifically 3CDpro T181K mutant)
OOutcomeProteolytic activity and RNA polymerase activitysurrogate
The study demonstrates that the poliovirus 3CD precursor possesses proteolytic activity but requires cleavage to 3Dpol to activate its RNA polymerase function.