Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.
The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.
Department of Biochemistry, University of Cambridge, United Kingdom.
A
A
Kaminski
S L
SL
Hunt
J G
JG
Patton
R J
RJ
Jackson
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002457:Cell Extracts; D002846:Chromatography, Affinity / Q000379:methods*; D016213:Cyclins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004686:Encephalomyocarditis virus / Q000235:genetics*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics; D004680:Maus Elberfeld virus / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D011072:Poly U / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012156:Reticulocytes / Q000737:chemistry; D012270:Ribosomes / Q000235:genetics; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002457:Cell Extracts; D016213:Cyclins; D004268:DNA-Binding Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D038941:Polypyrimidine Tract-Binding Protein; D011072:Poly U; D005982:Glutathione Transferase