Cleavage of Eukaryotic Translation Initiation Factor 4G by Exogenously Added Hybrid Proteins Containing Poliovirus 2A^pro in HeLa Cells: Effects on Gene Expression

Abstract

Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2A^pro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2A^pro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated.

ACKNOWLEDGMENTS

The expert technical assistance of M. A. Sanz is acknowledged. E. Feduchi, J. M. Sierra, and M. G. Rush are acknowledged for their help and for critical reading of the manuscript. We thank M. Cotten (Research Institute of Molecular Pathology, Vienna, Austria) for kindly providing CELO virus. H. Bujard (Zentrum für Molekulare Biologie, Heidelberg, Germany) is acknowledged for providing the HeLa cell line X1/5. N. Sonenberg is acknowledged for providing specific antibodies against human eIF4G-2.

Plan Nacional project PB94-0148 and the institutional grant to the CBM of Fundación Ramón Areces are acknowledged for financial support. I.N. is a holder of a Gobierno Vasco fellowship.