Internal ribosome entry mediated by a cellular or viral IRES is commonly studied by monitoring the synthesis of IRES-dependent reporter proteins. The poliovirus IRES can also be modified in its normal location in an mRNA, the viral genome, and the effects on the viral-replication cycle can then be determined in infected cells or organs. This combination affords a unique approach to the study of translational control in mammalian tissues.
Although the poliovirus receptor is produced in a broad range of organs (17
), viral replication and disease are limited to a few sites, including the brain, spinal cord, and alimentary tract. It has been proposed that this restriction is established at the step of IRES-mediated translation of the viral RNA (11
). This hypothesis cannot be tested in cell culture, because poliovirus replication occurs in cell lines derived from many organs that do not support viral replication (48
). However, direct measurements of viral IRES-mediated translation in organs would address the question of whether IRES activity correlates with the sites of virus replication in the animal. Previous analyses of transgenic mice that express a bicistronic mRNA have demonstrated a lack of organ specificity in translation mediated by the IRES of encephalomyocarditis virus (49
) and Theiler murine encephalomyelitis virus (50
). In the present study, the use of recombinant adenoviral vectors permitted the analysis of the IRES from multiple viruses in a fraction of the time required for a transgenic approach. Contrary to expectations, the IRESs of HCV, poliovirus, and CVB3 mediate translation in many murine organs, including those that are not sites of virus replication. Therefore, levels of IRES-dependent translation do not determine the organ-specific pattern of poliovirus replication. By expression of bicistronic mRNAs with adenovirus vectors, a pattern of IRES-mediated translation was revealed in organs that was not apparent in studies of cultured cells. It would therefore be of interest to use this approach to study the effect of different physiological states on cellular IRES-mediated translation.
Mouse models of human poliomyelitis may also provide insight into the relationship between poliovirus IRES–mediated translation and viral pathogenesis. Transgenic mice that produce the poliovirus receptor, CD155, are susceptible to poliovirus infection and develop disease that clinically and histopathologically resembles poliomyelitis (40
). Although an excellent animal model for poliomyelitis, CD155 transgenic mice are not susceptible to poliovirus infection by the oral route, the natural means of infection in humans (52
). We found that the tropism of recombinant polioviruses dependent on the IRES of CVB3 or HCV is unchanged in CD155 transgenic mice. These findings support the conclusion that a viral IRES can mediate translation in a wide range of organs and does not determine where poliovirus replicates and causes disease.
Previously, poliovirus dependent on the HCV IRES was reported to be cleared from the brain and spinal cord of adult mice without causing disease (13
). These results led to the conclusion that the HCV IRES does not mediate translation initiation in the murine brain and spinal cord. An alternative explanation is that the recombinant virus replicates poorly (53
) and is cleared by the immune system. We found that P1/HCV is cleared from adult CD155 transgenic mice but replicates in the brain and spinal cord of newborn CD155 transgenic mice and causes flaccid paralysis. Newborn mice are more susceptible than adults to infection with neurotropic viruses, including poliovirus (54
), but the basis for the increased susceptibility to poliovirus infection is not known. Taken together with the observation that the HCV IRES mediates translation in multiple organs, the finding that P1/HCV replicates and causes paralysis in newborn mice indicates that the murine brain and spinal cord produce the proteins required for HCV IRES–dependent initiation.
A recombinant poliovirus dependent on the human rhinovirus 2 IRES is neuroattenuated in adult mice (12
). A similar recombinant poliovirus is also neuroattenuated in newborn CD155 transgenic mice (data not shown). While the mechanism of attenuation is unclear, the HRV2 IRES may be dependent on cell proteins not present in the brain and spinal cord. This possibility is currently being explored.
Assertions that poliovirus tropism may be determined at the step of translation initiation (12
) were in part based on previous studies of the mechanism of attenuation of the poliovirus live vaccine strains. The C472U mutation within the IRES of the Sabin type 3 vaccine strain attenuates neurovirulence (23
) and was shown to cause a translation defect in vitro (28
). The Sabin type 3 vaccine replicates poorly in a neuroblastoma cell line but not in HeLa cells, a phenotype attributed to the C472U mutation (30
). Poor replication in neuroblastoma cells was believed to be a consequence of reduced IRES-mediated translation (27
). These observations led to the hypothesis that attenuating mutations in the Sabin vaccine IRES change poliovirus tropism by causing a translation defect specific to the brain and spinal cord (27
). To address this hypothesis, we measured IRES-mediated translation in organs and cells. Contrary to our expectation, the C472U mutation leads to identical translation defects in neuronal and non-neuronal organs and cells. This translation defect should lead to decreased viral replication in neuronal and non-neuronal organs. Our observation that the C472U mutation led to reduced IRES-mediated translation in all organs examined is consistent with the previous finding that this mutation reduces translational efficiency in extracts of non-neuronal cells (28
). This observation is not consistent with previous conclusions that the effect of the C472U mutation is specific for cells of neuronal origin and does not decrease viral translation or replication in HeLa cells (27
). In our study, variation in mRNA levels that could influence synthesis of the IRES-dependent reporter protein is controlled by the use of bicistronic mRNAs to quantify IRES-mediated translation. Furthermore, IRES-mediated translation might not be the rate-limiting step of viral replication in HeLa cells.
Mutations in the IRES are major determinants of the low pathogenicity of the poliovirus vaccine strains (22
). The results reported here indicate that the C472U attenuating mutation reduces IRES-dependent translation in all tissues, not only the CNS as previously hypothesized. Furthermore, we find that the C472U mutation does not eliminate viral replication in the CNS, since viruses with this mutation are neurovirulent in newborn mice. How might the C472 mutation reduce poliovirus neurovirulence in humans, yet still allow for sufficient virus replication to ensure protective immunity? Poliovirus infection of humans begins in the alimentary tract, and disease of the CNS is rare (58
). Replication defects in the alimentary tract associated with C472U may decrease the probability that a sufficient number of virus particles will reach the brain and spinal cord to initiate a productive infection, without impairing the immunogenicity of the vaccine. Testing this hypothesis will require the development of a transgenic mouse that is susceptible to poliovirus infection by the oral route.
Our results show that polioviruses with the C472U mutation are neurovirulent in newborn but not adult mice. The basis for this difference in pathogenicity is not known. One possibility is that the immature immune system of the newborn mouse cannot effectively clear the virus infection, even though viral replication is compromised by the C472U mutation. Our findings emphasize that attenuation of poliovirus neurovirulence is not simply a matter of whether or not IRES-mediated translation occurs in the brain and spinal cord. The outcome of infection is determined by a complex interplay between virus and host.