Abstract
The genomic RNA of encephalomyocarditis virus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins 2A and 2B by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3 (eRF1 and eRF3), aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site. We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs. Our results indicate that this unusual event occurs in the elongation phase of translation.
Highlights
The mechanism by which the encephalomyocarditis virus protein 2A-2B is processed into 2A and 2B is unknown
To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3, aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site
That system depended on the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), a cardiovirus of the picornavirus family, and it included purified eukaryotic translation initiation factors and the 40 S ribosomal subunit (7)
Summary
The mechanism by which the encephalomyocarditis virus protein 2A-2B is processed into 2A and 2B is unknown. That system depended on the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), a cardiovirus of the picornavirus family, and it included purified eukaryotic translation initiation factors (eIFs) and the 40 S ribosomal subunit (7). They subsequently reconstituted the initiation phase driven by the hepatitis C virus (HCV) IRES (8) and the cap structure (9). We reconstituted the HCV IRES-dependent protein synthesis system with eukaryotic elongation factor (eEF) 1, eEF2, eRF1, eRF3, aminoacyl-tRNA synthetases, tRNAs, and ribosomes and demonstrated that 2A-2B processing did not require eRFs
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