An improved method for detection and quantification of differential interactions between poliovirus internal ribosome entry site RNA and pyrimidine tract binding protein from primary cells.
Attenuated and pathogenic viral variants are often extremely similar viruses with drastically different replication potentials. Despite precise knowledge of viral residues responsible for poliovirus attenuation/neurovirulence, molecular mechanisms mediating these effects remain poorly understood. Data from numerous sources suggest that a functional difference in translation initiation is one responsible factor. However, direct evidence, as well as a comprehensive model are lacking. Several difficulties, including lack of an assay system to quantify differential internal ribosome entry site/pyrimidine tract binding protein interaction in relevant systems, have precluded progress. A novel assay system that overcomes some difficulties is presented below. The assay uses streptavidin paramagnetic particles, biotinylated RNA and glutathione-S-transferase/pyrimidine tract binding protein fusion to detect nanogram levels of uncloned cellular pyrimidine tract binding protein species that interact with internal ribosome entry site RNA. Using this assay, it was shown that pyrimidine tract binding protein from primary human monocytes binds to internal ribosome entry site RNA from virulent poliovirus better than to that from attenuated virus, while pyrimidine tract binding protein from HeLa cells does not distinguish between the two internal ribosome entry sites. Since primary human monocytes reflect neurovirulence-related differential poliovirus replication, these results suggest that pyrimidine tract binding protein may contribute to differential poliovirus replication in vivo. This assay also has the potential to be applicable broadly to other nucleic acid/protein interactions.
Miles
D H
DH
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112, USA.
Johnston
K H
KH
Freistadt
M S
MS
D020121:5' Untranslated Regions / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism / Q000821:virology; D006801:Humans; D009000:Monocytes / Q000378:metabolism / Q000821:virology; D011051:Poliomyelitis / Q000821:virology*; D017955:Poliovirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D038941:Polypyrimidine Tract-Binding Protein; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014773:Virology / Q000379:methods
D020121:5' Untranslated Regions; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein