Initiation of translation of the single-stranded genomic RNAs of picornaviruses such as poliovirus (PV) and encephalomyocarditis virus (EMCV) is cap-independent and controlled by a long segment within the 5' non-translated region (5'NTR), termed internal ribosomal entry site (IRES). Cellular RNA-binding proteins have been identified that are involved in IRES function in trans. One of these proteins (p57) has been found to be identical to the polypyrimidine tract binding protein (pPTB), a nuclear protein implicated in various processes involving pre-mRNA. Anti-pPTB antibodies inhibit picornavirus mRNA, but not globin mRNA translation, in vitro. Proof for the 5'-independent initiation of translation in vivo was obtained by inserting the EMCV IRES into the ORF of PV thereby constructing a dicistronic, viable poliovirus with the genotype [PV] 5'NTR-P1-[EMCV] IRES-[PV] P2-P3-3'NTR. Dicistronic polioviruses were also constructed that served as novel expression vectors where a foreign gene has been inserted into the PV genome. Incubation of poliovirus RNA in a HeLa cell-free extract leads to the synthesis and processing of viral proteins, viral RNA replication followed by formation of infectious virions. Cell-free synthesis of PV has nullified the dictum that no virus can multiply in a cell-free medium. The genome replication of poliovirus and the mechanism of recombination in poliovirus replication is still not fully understood. Biochemical evidence has been obtained that the conserved NTP-binding motif in PV protein 2C is essential for RNA replication and virus propagation. Finally by using genetic studies we found that during viral RNA synthesis a poliovirus containing two tandemly arranged VPgs (3A-VPg1-VPg2-3Cpro) led to the removal of the 3C-proximal VPg copy.