Mutational analysis of poliovirus 2Apro. Distinct inhibitory functions of 2apro on translation and transcription.
Transient expression of poliovirus 2Apro in mammalian cells by means of the recombinant vaccinia virus vT7 expression system leads to drastic inhibition of both cellular and vaccinia virus gene expression (Aldabe, R., Feduchi, E., Novoa, I., and Carrasco, L. (1995) FEBS Lett. 377, 1-5; Aldabe, R., Feduchi, E., Novoa, I., and Carrasco, L. (1995) Biochem. Biophys. Res. Commun. 215, 928-936). To obtain further insights into the molecular basis of this inhibition, a number of 2Apro variants were generated and expressed in COS-1 cells. The effect of these variants on cellular translation, on vaccinia virus-specific translation, and on transcription of the reporter gene luciferase was analyzed. The ability of the different 2Apro variants to block cellular translation depends on their capacities to cleave eIF-4G. The blockade exerted by 2Apro on transcription of the luciferase gene reinforces the notion that this protease is a potent inhibitor of RNA polymerase II-mediated transcription. Some of the 2Apro variants tested failed to block luciferase transcription, despite the fact that eIF-4G cleavage and inhibition of translation were observed. Two reconstituted polioviruses mutated in 2Apro were defective in inhibiting luciferase transcription, yet were still able to cleave eIF-4G and block translation. These findings indicate that 2Apro interferes with cellular gene expression at both the transcriptional and translational levels. Moreover, these two effects probably reflect the inactivation of different host proteins by poliovirus 2Apro.
Ventoso
I
I
Centro de Biología Molecular (Consejo Superior de Investigaciones Científicas), Universidad Autónoma de Madrid, Canto Blanco, 28049 Madrid, Spain. iventoso@trasto.cbm.uam.es
Barco
A
A
Carrasco
L
L
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D017354:Point Mutation; D017955:Poliovirus / Q000201:enzymology*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis
D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D008156:Luciferases; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus