Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein

Abstract

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.

Cell-specific proteins regulate viral RNA translation and virus-induced disease

“…PCBP is ubiquitously expressed, while, as demonstrated in the present study, nPTB substitutes for the function of PTB in neurons. The attenuation mutation in poliovirus type 3 reduced cross‐linking of a fragment of the poliovirus 5′ UTR to PTB in neuroblastoma cells but not in HeLa cells (Gutierrez et al ., 1997). It may be that PTB binds to the IRES of Sabin strains more stably than nPTB, thereby allowing for better translation and growth in non‐neural cells compared with those in neurons.…”

Polypyrimidine tract‐binding protein interacts with HnRNP L

“…The role played by PTB in IRES‐dependent translation has been investigated. Mutational analyses have revealed a strong correlation between the binding affinity of PTB to the picornaviral IRESs and the translational efficiency of the corresponding mRNAs [8, 11, 14]. Depletion of endogenous PTB from HeLa cell lysates and rabbit reticulocyte lysates (RRL) selectively prevented the EMCV IRES‐dependent translation [13].…”

Human astroviruses: in silico analysis of the untranslated region and putative binding sites of cellular proteins

“…1a) are present only in classical viruses. Additionally, another sequence is conserved between the former motifs, TGGT residues are common in all classical HAstV (position nt [13][14][15][16], except in HAstV-6 where the last T residue changes to C, as well in VA and AST/PS genotypes. In MLB 1-3 clade viruses (position nt 8-11) as well in VA1-4 clade virus this motif is present but in different position (underlined).…”

“…For example, heterogeneous nuclear ribonucleoproteins (hnRNPs), such as polypyrimidine tract-binding protein (PTB), also known as hnRNP1, is an RNA binding protein involved in multiple aspects of cellular mRNA metabolism, including splicing regulation, polyadenylation, 3′ end formation, translation of RNAs, RNA localization and stability. Additionally, PTB binding to viral RNAs is by far the best characterized to date [14,21,24,25,41,49,50,53,54].…”