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Biochemistry. 1997 Jun 24;36(25):7802-9.

Cleavage of p220 by purified poliovirus 2A(pro) in cell-free systems: effects on translation of capped and uncapped mRNAs.

Author information

1
Centro de Biología Molecular, UAM-CSIC, y Centro Nacional de Biotecnología, CSIC, Universidad Autónoma de Madrid, Cantoblanco, Spain.

Abstract

Poliovirus protease 2A(pro) has been obtained in soluble form as a fusion protein with maltose binding protein (MBP). Addition of MBP-2A(pro) to rabbit reticulocyte cell-free systems gives rise to efficient cleavage of the initiation factor of translation p220 (eIF-4G). Translation of capped mRNA encoding the influenza virus NP protein is severely impaired in lysates in which p220 has been proteolytically cleaved. This inhibition is dependent on the concentration of mRNA added to the lysate. Thus, increasing the concentrations of mRNA substantially overcomes the blockade of NP synthesis after p220 cleavage. Notably, translation of uncapped NP mRNA is also compromised in p220-deficient rabbit reticulocyte lysates, suggesting that p220 participates in the translation of both capped and uncapped NP mRNAs. The effects of p220 proteolysis by poliovirus 2A(pro) have also been assayed on luciferase mRNA translation. Three types of mRNAs encoding for luciferase have been examined: capped, uncapped, and mRNA bearing the poliovirus 5' leader region (leader luc mRNA). Synthesis of luciferase directed by any of these mRNAs was inhibited after cleavage of p220 in rabbit reticulocyte lysates. Interestingly, supplementation of the lysate with HeLa cell extracts stimulates leader luc mRNA translation by poliovirus 2A(pro). These results indicate that activation of translation of mRNAs bearing the poliovirus leader region promoted by this poliovirus protease requires a factor present in HeLa cell extracts. These findings agree well with recent experiments implicating p220 not only in protein synthesis directed by capped mRNAs but also in the translation of naturally uncapped mRNAs.

PMID:
9201923
DOI:
10.1021/bi9631172
[Indexed for MEDLINE]

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