Variations in cap-binding complexes from uninfected and poliovirus-infected HeLa cells.
Poliovirus infection of HeLa cells results in cleavage of the p220 subunit of eukaryotic initiation factor eIF-4F and inhibits cap-dependent initiation of protein synthesis. To examine the effect of virus-induced inhibition on the structure of initiation factor complexes involved in cap binding, the polypeptide compositions of cap affinity-purified complexes from uninfected and poliovirus-infected HeLa cells were analyzed. Monoclonal antibodies directed against p220 and an eIF-3 subunit, p170, were utilized to locate eIF-3 and eIF-4F on sucrose gradients and in fractions eluting from cap analog columns. This approach resulted in the purification of several different cap-binding complexes from different cellular subfractions and revealed significant differences in their composition after infection. The results indicate that eIF-3 and eIF-4F bind to the cap structure, possibly in the form of a complex, and that a modified form of eIF-3 alone has some cap-binding activity in the complete absence of p220, eIF-4A, and eIF-4E. Ribosome-derived complexes containing cleaved p220 are no longer associated with eIF-3 or eIF-4A, and a significant amount of cleaved p220 is associated with a unique cytoplasmic cap-binding complex. The cytoplasmic complex also contains Mr = 170,000 and 80,000 polypeptides, neither of which are major components of eIF-4F. These results demonstrate significant variation in the composition of cap-binding complexes from both infected and uninfected cells. They indicate that eIF-3 might play a direct role in cap binding and suggest that poliovirus-induced cleavage of p220 results in the release of the eIF-4A subunit from eIF-4F and abolishes an association between eIF-4F and eIF-3 which may function during the multifactor steps involved in initiation of cap-mediated translation.
Etchison
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Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66103.
Smith
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D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D039562:Eukaryotic Initiation Factor-4F; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000378:metabolism*; D017955:Poliovirus / Q000235:genetics*; D039381:RNA Cap-Binding Proteins; D012270:Ribosomes / Q000378:metabolism
D002352:Carrier Proteins; D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D039381:RNA Cap-Binding Proteins