Assuntos
Humanos , Masculino , Feminino , Virologia/classificação , Virologia/instrumentação , Virologia/métodosAssuntos
Humanos , Masculino , Feminino , Virologia/classificação , Virologia/instrumentação , Virologia/métodosAssuntos
Masculino , Feminino , Humanos , Virologia/classificação , Virologia/instrumentação , Virologia/métodosAssuntos
Masculino , Feminino , Humanos , Virologia/classificação , Virologia/instrumentação , Virologia/métodosRESUMO
Mutations in the 5' untranslated regions (5'-UTRs) of all three serotypes of the Sabin vaccine strains are known to be major determinants of the attenuation phenotype. To further understand the functional basis of the attenuation phenotype caused by mutations in the 5'-UTR, we studied their effects on viral replication, translation, and the interaction of the viral RNA with cell proteins. A mutation at base 472 (C472U), which attenuates neurovirulence in primates and mice, was previously found to reduce viral replication and translation in neuroblastoma cells but not in HeLa cells. This mutation reduced cross-linking of the poliovirus 5'-UTR to polypyrimidine tract-binding protein (pPTB) in neuroblastoma cells but not in HeLa cells. These defects were absent in a neurovirulent virus with C at nucleotide 472. When C472U and an additional mutation, G482A, were introduced into the 5'-UTR, the resulting virus was more attenuated, had a replication and translation defect in both HeLa cells and neuroblastoma cells, and cross-linked poorly to pPTB from both cell types. A neurovirulent revertant of this virus (carrying U472C, G482A, and C529U) no longer had a replication defect in HeLa and SH-SY5Y cell lines and cross-linked with pPTB to wild-type levels. The results suggest that the attenuating effects of the mutation C472U may result from an impaired interaction of the 5'-UTR with pPTB in neural cells, which reduces viral translation and replication. Introduction of a second mutation, G482A, into the 5'-UTR extends this defect to HeLa cells.
Assuntos
Poliovirus/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Células HeLa , Humanos , Mutação , Fenótipo , Poliovirus/fisiologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Biossíntese de ProteínasRESUMO
The restricted tissue tropism observed in poliovirus infection is not governed solely by the expression of the poliovirus receptor (PVR) gene, but might be controlled at stages beyond virus entry, such as translation, replication, or assembly. Translation of poliovirus RNA by a cap-independent mechanism requires interactions of the 5'-untranslated region (5'UTR) with cell proteins. To determine whether the patterns of these interacting proteins differ in HeLa cells and permissive and nonpermissive tissues, UV-crosslinking assays using the poliovirus 5'UTR and tissue extracts from PVR transgenic mice were performed. The results indicate a correlation between the presence of a 97-kDa UV-crosslinked protein and permissivity to poliovirus infection. Acquired poliovirus susceptibility in in vitro-cultured kidney cells also correlates with the presence of a 97-kDa crosslinked band. The interaction of the 97-kDa protein from HeLa cells and mouse brain with the poliovirus 5'UTR is stable and specific. Whether the 97-kDa protein plays a role in poliovirus translation and tissue susceptibility remains to be determined.