Molecular Mechanisms of Attenuation of the Sabin Strain of Poliovirus Type 3

Abstract

Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES).We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on the Sabin3 IRES than on the PV3 IRES. Translation mediated by the PV3 and Sabin3 IRESs in neurons of the chicken embryo spinal cord demonstrated a translation deficit for the Sabin3 IRES that could be rescued by increasing PTB expression in the CNS. These data suggest that the low levels of PTB available in the CNS, coupled to a reduced binding of PTB on the Sabin3 IRES, leads to its CNS-specific attenuation. This study also demonstrates the use of the chicken embryo to easily investigate translation of RNA within a neuron in the CNS of an intact living organism.Poliovirus (PV) infection worldwide has been successfully controlled through the use of two highly effective vaccines. The first, developed by Salk and colleagues, was prepared by formalin treatment of strains from the three major serotypes of PV to generate a killed-virus vaccine. The second, a live, attenuated virus vaccine developed by Sabin and colleagues, was prepared by serial passage of the three major PV strains in nonhuman primates and in cultured primate cells until strains with reduced neurovirulence were obtained (46). These Sabin strains are significantly attenuated in the central nervous system (CNS) but replicate at wild-type (WT) levels in the gut; similarly, these strains do not grow as well in cultured neuroblastoma cells as they do in cultured HeLa cells (25,26). The Sabin strains are therefore considered to have a neural cellspecific attenuation.PV has a single-stranded, positive-sense RNA genome of greater than 7,500 nucleotides (nt). An internal ribosome entry site (IRES) located in the 5Ј untranslated region (UTR) of the picornavirus RNA genome mediates efficient translation initiation of the viral polyprotein (reviewed in references 18 and 41). Studies of recombinant PVs made between attenuated vaccine strains and their neurovirulent progenitors have shown that a major determinant of neuroattenuation maps to a single point mutation located within the viral IRES at nt 480, 481, or 472 in the case of Sabin type 1 (Sabin1), Sabin2, and Sabin3, respectively (21,35,49). In addition, the Sabin3 vaccine strain has been found to revert in humans to neurovirulence, and this reversion is associated with a mutation that restores the WT sequence at nt 472 (6). Studies have demonstrated a translational deficiency of the Sabin3 IRES in Krebs-2 cells and neuroblastoma cells that is not present in HeLa cells (26,47,48). These findings have led to the proposal that the attenuating point mutation located in the IRES of the Sabin vaccine strains inhibits IRES-medi...

Cell-Type-Specific Repression of Internal Ribosome Entry Site Activity by Double-Stranded RNA-Binding Protein 76

“…The IRES stimulatory activity of PTB was demonstrated in vivo by transient overexpression (16) and targeted depletion (14). In addition, variable IRES trans activity of PTB and its neural isoform nPTB have been implicated in the neuroattenuation phenotype of the serotype 3 Sabin strain (22). Cell-type-specific growth of the picornavirus hepatitis A virus is determined by the relative abundance of PTB, a stimulatory ITAF, and glyceraldehyde 3-phosphate dehydrogenase, an inhibitory ITAF (46), demonstrating that cell-typespecific viral propagation can be regulated by IRES-binding proteins with opposing effects on translation.…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…Noteworthy, domain IV harbors a GNRA motif (N stands for any nucleotide, and R, purine) (Bailey and Tapprich, 2007;Du et al, 2004) and the C-rich loop, also conserved in type II IRES elements. Domain V plays a critical role in PV life cycle (Guest et al, 2004). This stem-loop harbors a major determinant of PV neurovirulence, as indicated by the mutation at position 472 found in Sabin 3 strains recovered from vaccinated patients that developed poliomyelitis (Evans et al, 1985).…”

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation

“…Poliovirus IRES activity is enhanced by La protein (Meerovitch et al 1993), poly(rC)-binding protein 2 (PCBP2) (Blyn et al 1997), and the protein encoded by the gene upstream of N-ras (UNR) (Boussadia et al 2003), as well as by PTB (Gosert et al 2000). In the brain, lower levels of PTB may contribute to the attenuation of the poliovirus Sabin strains (Guest et al 2004) in addition to the impaired binding of initiation factors (Ochs et al 2003). Rhinovirus IRES activity responds to the synergistic action of UNR and PTB (Hunt et al 1999).…”