Key Points
- To determine the intracellular localization of poliovirus protease 2Apro and evaluate its regulatory effects on host and viral mRNA translation in mammalian cells.
- Transfected HeLa and COS mammalian cells with plasmid pTM1-2A and infected them with recombinant vaccinia virus expressing T7 RNA polymerase.
- Tracked the intracellular localization of 2Apro and monitored cleavage kinetics of translation initiation factor eIF-4F component p220.
- Assessed protein synthesis of vaccinia virus, vesicular stomatitis virus, and an EMC untranslated leader-driven poliovirus 2C construct.
- Expressed poliovirus 2Apro localized to the cytoplasm, primarily within the endoplasmic reticulum and scattered cytoplasmic vesicles.
- Selective cleavage of p220 occurred starting at 5 hours post-infection, directly coinciding with the profound inhibition of vaccinia and vesicular stomatitis virus protein translation.
- Synthesis of poliovirus protein 2C driven by a picornavirus untranslated leader region continued unimpeded despite 2Apro expression.
Structured PICO
PPopulationHeLa and COS cells
IInterventionExpression of poliovirus protease 2Apro via transfection with vector pTM1-2A and infection with recombinant vaccinia virus bearing T7 RNA polymerase
OOutcomeCleavage of p220 and effects on translation of viral proteins
The study demonstrates that poliovirus 2Apro cleaves p220, inhibiting capped mRNA translation while sparing translation driven by a picornavirus leader region.