Poliovirus proves IRES-istible in vivo

The attenuation of poliovirus, the prototype member of the virus family Picornaviridae, by Albert Sabin’s multiple, alternating passages of all three serotypes in different cultured cells and host animals led to the isolation of the vaccine strains now in use throughout much of the world. The nature of the genetic differences among the attenuated (avirulent) isolates of poliovirus was unknown until the first complete genomic sequences of viral RNA were determined (1–4) and compared with the sequences of different Sabin strain isolates (4–6). Depending on the serotype, there were nucleotide differences scattered throughout the genome or concentrated in just a few loci in the Sabin strain viral RNAs. As discussed in the comprehensive review of poliovirus genetics by Wimmer and colleagues (7), the most functionally significant differences contributing to the attenuation phenotype of the Sabin vaccine strains of poliovirus were found in the genomic RNA sequences corresponding to the 5′ noncoding region (5′ NCR) and to sequences that encoded the capsid proteins, which make up the icosahedral shell of this nonenveloped virus particle. The genome organization of poliovirus RNA is depicted in Figure ​Figure1,1, while Figure ​Figure22 displays the RNA secondary structure of the 5′ NCR. Figure 1 Functional map of the poliovirus genome. Genomic RNA is linked to a virus-encoded peptide (VPg) at the 5′ end and a genetically coded poly(A) tract at the 3′ end. Viral RNA is depicted with a description of the functions of the various ... Figure 2 Predicted RNA secondary structure of the poliovirus 5′ NCR. Computer prediction and chemical and enzymatic RNA-structure probing were used to deduce a consensus RNA conformation. Conserved sequences among picornaviruses include a GNRA tetraloop ... A major determinant of neurovirulence for all three serotypes of poliovirus was identified in the stem-loop V region of the 5′ NCR, indicated by the star in Figure ​Figure22 (ref. 8; for recent reviews, see refs. 9, 10,). This stem-loop structure was later shown to be part of the internal ribosome entry site (IRES) present in the 5′ NCRs of all picornavirus positive-strand genomic RNAs (Figure ​(Figure2).2). IRES elements permit the internal binding of ribosomes via a non-canonical, cap-independent mechanism of translation initiation utilized by some viruses and even a limited number of eukaryotic cellular mRNAs. There is some evidence for cell type–specific, cell cycle–dependent, and even developmentally regulated translation initiation mediated by cellular IRES elements (11). Thus, it would certainly be reasonable to assume that viral IRES elements, like the one encoded in the poliovirus genome, might have cell-specific requirements to achieve high levels of virus-specific translation initiation. Given the identification of a major attenuation determinant in an important RNA secondary structure embedded in the poliovirus IRES, this conclusion might appear foregone. Indeed, the analysis of attenuated and neurovirulent polioviruses using cell culture and in vitro translation assays provided considerable evidence for the role of the IRES in the biological properties of these viruses. RNA isolated from Sabin type 3 poliovirus was translated in vitro with reduced efficiency compared with RNAs from neurovirulent strains of type 3 poliovirus (12). The translation deficiency was later shown to be the result of the known attenuating mutation (N472 C O U) in the IRES of the Sabin type 3 genome (13). A cell culture model using HeLa cells and a neuroblastoma cell line showed that recombinant viruses differing only by the C472U mutation replicated with equal efficiency in HeLa cells (14). However, infection of neuroblastoma cells with these two viruses showed that the virus containing the uridine residue at N472 grew to lower titers in these cells. The attenuated virus also had reduced translation efficiency during infection of the neuronal cell line, consistent with the above-noted in vitro translation results for Sabin 3 poliovirus RNAs. In addition, site-directed mutations that altered the RNA sequences and secondary structures adjacent to the major attenuation determinant in the 5′ NCR of type 1 poliovirus (N480) led to neuronal cell–specific growth defects in cultured cells and to in vitro translation defects in S-10 extracts derived from neuroblastoma cells grown in suspension culture (15). As they report in this issue of the JCI, Kauder and Racaniello formally tested the hypothesis that IRES-mediated translation initiation is a major determinant of poliovirus tropism and pathogenesis (16).