Poliovirus (PV) translation and replication can occur in neuronal cells where it causes degeneration and lysis of cells leading to paralytic poliomyelitis. Other cell types are much less affected by PV infection and do not support translation and replication of the virus as well. Apart from the poliospecific receptor, the reasons for the tissue preference of poliovirus may be found in its translation initiation via an internal ribosome entry site (IRES), which in addition to cellular initiation factors uses proteins normally not involved in translation to achieve efficient translation (ITAFs). Several ITAFs are known, but neither do these known factors explain the tissue specificity of poliovirus nor are they sufficient to initiate translation at the PV IRES using only purified components. Additional factors may be present in cells, especially in neuronal cells, that are both necessary for translation initiation on the PV IRES. One potential new factor, ErB3 binding protein (EbP1), was identified here to bind to the PV IRES. Translation initiation complexes were formed with labelled Poliovirus IRES RNA in an in vitro reconstitution system. A double labeled RNA allowed to identify 48S complexes in sucrose gradients and to enrich proteins present in these complexes. As controls RNAs containing the IRESs of Hepatitis C virus (HCV) and encephalomyocarditis virus (EMCV) were used. One protein, specific for PV and EMCV was identified in the light RNA peak but not in the 48S peak. It was identified by mass spectrometry as EbP1, which is a known ITAF for the IRES of Foot-and-mouth-disease virus. However, its role in PV translation initiation seems to be inhibiting rather than stimulating since it could not be found in 48S complexes. Additionally I identified a neuron specific microRNA (miRNA) that stimulates translation at the Poliovirus IRES. By screening the poliovirus IRES sequence for potential target sites for miRNAs that are preferentially expressed in neuronal cells, I found sequences partially complementary to three neuron-specific miRNAs, miRNA-127, miRNA-326 and miRNA-422a. These miRNAs were co-transfected into cultured cell-lines together with a poliovirus IRES reporter RNA. Of these miRNAs, only miRNA-326 enhanced poliovirus translation efficiency. The target site for miRNA-326 is located in a loop of the central IRES domain IV. Mutagenesis of this miRNA-326 target site disabled the stimulation by added miRNA-326, indicating a physical interaction of miRNA-326 with the poliovirus IRES. However, this mutation could only be partially rescued by a miRNA with a complementary mutation in the seed sequence of miRNA-326. Since two binding sites of miRNA-326 in the 5 untranslated region of PV are identical with binding sites for the known ITAF PCBP2, a connection between those two factors may be assumed.
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