A Translation System Reconstituted with Human Factors Proves That Processing of Encephalomyocarditis Virus Proteins 2A and 2B Occurs in the Elongation Phase of Translation without Eukaryotic Release Factors

作者：Machida Kodai; Mikami Satoshi; Masutani Mamiko; Mishima Kurumi; Kobayashi Tominari; Imataka Hiroaki^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289(46): 31960-31971.

DOI：10.1074/jbc.M114.593343

• Protein Expression
• Protein Processing
• Protein Synthesis
• Translation Regulation
• Viral Protein
• Cell-free System
• Reconstitution System

Background: The mechanism by which the encephalomyocarditis virus protein 2A-2B is processed into 2A and 2B is unknown. Results: A translation system reconstituted with defined human factors recapitulates 2A-2B processing. Conclusion: 2A-2B processing occurs at the elongation step. Significance: The translation system reconstituted with human factors should be a useful tool for studying eukaryotic protein synthesis mechanisms. %26lt;br%26gt;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.

• 出版日期2014-11-14
• 单位RIKEN