Mutational Analysis of Poliovirus 2Apro

Ventoso, Iván; Barco, Ángel; Carrasco, Luís

doi:10.1074/jbc.273.43.27960

Cited by 35 publications

(36 citation statements)

References 51 publications

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“…Thus, hydrolysis of eIF4GI occurs after eIF4GII cleavage. This method is much more efficient, rapid, and reliable than other methods previously described, such as HeLa cell lines that can be induced to express PV 2A pro , direct penetration of hybrid proteins that contain this protease, or coupled infection with recombinant vaccinia virus T7 and transfection with pTM1-2A (20,17,21).…”

Section: Discussionmentioning

confidence: 99%

“…Plasmids-The plasmids pKs-Luc, pTM1-Luc, pTM1-2C, and pTM1-2A and the plasmids pTM1-2AG60R, pTM1-2AG121E, pTM1-2AD135N, and pTM1-2AV119M, which encodes a PV 2A pro mutant, have been described previously (20,21).…”

Section: Methodsmentioning

confidence: 99%

“…Two PV 2A pro variants, D135N (M6) and V119M (M7), that failed to repress host transcription but maintained their capacity to proteolyze eIF4GI, have been described (21). Because pro M6 and M7 failed to repress host transcription (supplemental Fig.…”

Section: Involvement Of Eif4gi and Eif4gii In The Translation Ofmentioning

confidence: 99%

“…The IRES region from EMCV confers a high translability to the mRNAs, and so transfection of EMC-2A could compete with cellular mRNAs, leading to the inhibition of host translation. To test this possibility, HeLa cells were transfected with mRNAs that encode for inactive point mutants of PV 2A pro (M2, G60R; M15, G121E) (21). These mRNAs did not affect the level of host protein synthesis as compared with control cells, and both isoforms of eIF4G remained intact at 8 h post-electroporation (hpe) (Fig.…”

Section: Transfection Of Hela Cells With Mrnasmentioning

confidence: 99%

“…Containing the PV 2A Sequence-We initially constructed a plasmid that encoded for the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) followed by the PV 2A pro sequence (pTM1-2A) (21). In vitro transcription from this plasmid leads to the synthesis of EMC-2A mRNA.…”

Section: Transfection Of Hela Cells With Mrnasmentioning

confidence: 99%

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Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

Castelló

Álvarez

Carrasco

2006

Journal of Biological Chemistry

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Two isoforms of the translation initiation factor eIF4G, eIF4GI and eIF4GII, have been described in eukaryotic cells. The exact function of each isoform during the initiation of protein synthesis is still under investigation. We have developed an efficient and reliable method of expressing poliovirus 2Apro, which differentially proteolyzes eIF4GI and eIF4GII in a timeand dose-dependent manner. This system is based on the electroporation of an in vitro transcribed mRNA that contains the encephalomyocarditis virus internal ribosome entry site followed by the sequence of poliovirus 2Apro. In contrast to HeLa cells, expression of this protease in BHK-21 cells induces delayed hydrolysis kinetics of eIF4GI with respect to eIF4GII. Moreover, under these conditions the polyadenylate binding protein is not cleaved. Interestingly, translation of de novo synthesized luciferase mRNA is highly dependent on eIF4GI integrity, whereas ongoing translation is inhibited at the same time as eIF4GII cleavage. Moreover, reinitiation of a preexisting mRNA translation after polysome run-off is dependent on the integrity of eIF4GII. Notably, de novo translation of heat shock protein 70 mRNA depends little on eIF4GI integrity but is more susceptible to eIF4GII hydrolysis. Finally, translation of an mRNA containing encephalomyocarditis virus internal ribosome entry site when the two isoforms of eIF4G are differentially hydrolyzed has been examined.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Involvement Of Eif4gi and Eif4gii In The Translation Ofmentioning

confidence: 99%

Section: Transfection Of Hela Cells With Mrnasmentioning

confidence: 99%

Section: Transfection Of Hela Cells With Mrnasmentioning

confidence: 99%

See 3 more Smart Citations

Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

Castelló

Álvarez

Carrasco

2006

Journal of Biological Chemistry

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Coding Changes in the Poliovirus Protease 2A Compensate for 5′NCR Domain V Disruptions in a Cell-Specific Manner

et al. 2000

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Polioviruses are single-stranded RNA viruses with an unusually long noncoding region (NCR) at the 5' end predicted to have an elaborate secondary structure made up of six domains. Mutations in domain V of the poliovirus 5'NCR that disrupt secondary structure are responsible for attenuation of the virus and a temperature-sensitive (ts) phenotype in vitro. In addition to direct back mutation or compensatory second site mutation in the 5'NCR as previously documented, the ts phenotype was found to be compensated for in monkey kidney cells in vitro by a coding change in the protease 2A. These coding changes were found throughout the protease with no obvious pattern or trend. They were not all found to be equivalent and limited in ability to compensate for the severest domain V disruption. The compensatory effect of the 2A changes was found to be cell specific, having no effect on monkey neurovirulence and in a mouse cell line but a significant effect in two monkey cell lines and a human epithelial line.

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Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli

et al. 2013

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A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C(pro)) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C(pro) partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C(pro). The EV71 3C(pro) had a Km value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C(pro) was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C(pro).

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Mutational Analysis of Poliovirus 2Apro

Cited by 35 publications

References 51 publications

Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

Coding Changes in the Poliovirus Protease 2A Compensate for 5′NCR Domain V Disruptions in a Cell-Specific Manner

Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli

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