Key Points
- To identify and map the primary initiation site for protein translation on foot-and-mouth disease virus RNA using defined RNA cleavage fragments.
- Translated intact viral RNA and defined RNA fragments in an mRNA-dependent reticulocyte lysate cell-free system.
- Cleaved viral RNA at the internal poly(C) tract using RNase H to produce distinct 5' (S) and 3' (L) fragments, with and without the 5'-terminal VPg protein.
- Labeled nascent initiation peptides using N-formyl [35S]methionine tRNAfMet to identify the polypeptide closest to the start site.
- The 3' L fragment directed the synthesis of the primary viral polypeptides P16, P20a, and P88 matching full-length RNA, while the 5' S fragment produced no defined translation products.
- Enzymatic removal of the 5'-terminal VPg protein produced no observable change in translation efficiency or synthesized protein profiles.
- N-formyl [35S]methionine tRNAfMet selectively labeled polypeptide P16 in full-length and L fragment translations, establishing that the major translation initiation site is situated 3' of the poly(C) tract.
Structured PICO
PPopulationIn vitro translation system (mRNA-dependent reticulocyte lysate) and foot-and-mouth disease virus RNA
IInterventionTranslation of defined fragments of the RNA (L and S fragments produced by RNase H digestion) and removal of VPg
CComparatorTranslation of full-length foot-and-mouth disease virus RNA
OOutcomeIdentification of the initiation site for protein synthesis and resulting polypeptide productssurrogate
This study maps the major initiation site for protein synthesis on the foot-and-mouth disease virus RNA to the 3' side of the poly(C) tract.