Purification of two picornaviral 2A proteinases: interaction with eIF-4 gamma and influence on in vitro translation.
A mammalian cell infected with a human rhinovirus or enterovirus has a much reduced capability to translate capped mRNAs (the host cell shutoff), while still allowing translation of uncapped viral RNA. Biochemical and genetic evidence suggests that the viral proteinase 2A induces cleavage of the eukaryotic initiation factor (eIF) 4 gamma (also known as p220) component of eIF-4 (formerly called eIF-4F). However, neither the mechanism underlying the specific proteolysis of eIF-4 gamma nor the influence of this cleavage on the translation of capped mRNAs has been clarified. Such studies have been hampered by a lack of large quantities of a purified 2A proteinase. Therefore, the mature proteinases 2A of human rhinovirus 2 and coxsackievirus B4 were expressed in soluble form in Escherichia coli. A four-step purification protocol was developed; 1 mg of highly purified 2A proteinase per gram wet weight of E. coli was obtained. Both enzymes cleaved directly eIF-4 gamma as part of the purified eIF-4 complex. Addition of HRV2 2A proteinase to HeLa cell cytoplasmic translation extracts resulted in eIF-4 gamma cleavage and drastically reduced the translation of capped mRNA; addition of purified eIF-4 restored translation to the initial level. However, translation of a reporter gene driven by the 5'-untranslated region of human rhinovirus 2 was translated 2-3-fold more efficiently in the presence of HRV2 2A proteinase.
Liebig
H D
HD
Institute of Biochemistry, Medical Faculty, University of Vienna, Austria.
Ziegler
E
E
Yan
R
R
Hartmuth
K
K
Klump
H
H
Kowalski
H
H
Blaas
D
D
Sommergruber
W
W
Frasel
L
L
Lamphear
B
B
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D039562:Eukaryotic Initiation Factor-4F; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010849:Picornaviridae / Q000201:enzymology*; D010957:Plasmids; D014176:Protein Biosynthesis / Q000187:drug effects*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D014764:Viral Proteins
D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins; C075833:eIF-4; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus