Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus.
The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4 gamma. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4 gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease. In this report we have used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4 gamma in vitro in the eIF-4 complex purified from rabbit reticulocytes. Neither the rate of cleavage nor fragment sizes were affected by addition of eIF-3. The NH2- and COOH-terminal fragments of eIF-4 gamma were separated by reverse phase HPLC and identified with specific antibodies, and the NH2-terminal sequence of the COOH-terminal fragment was determined by automated Edman degradation. The cleavage site for both proteases is 479GRPALSSR decreases GPPRGGPG494 in rabbit eIF-4 gamma, corresponding to 478GRTTLSTR decreases GPPRGGPG493 in human eIF-4 gamma.
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130-3932.
B J
BJ
Lamphear
R
R
Yan
F
F
Yang
D
D
Waters
H D
HD
Liebig
H
H
Klump
E
E
Kuechler
T
T
Skern
R E
RE
Rhoads
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004770:Enterovirus / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012229:Rhinovirus / Q000378:metabolism*; D014764:Viral Proteins
D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C077382:eIF-4gamma; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus