Genetic analysis of a poliovirus/hepatitis C virus chimera: New structure for domain II of the internal ribosomal entry site of hepatitis C virus

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses d irect cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric vi ral genomes in which IRES elements were exchanged from one virus to another . Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a H epacivirus belonging to Flaviviridae, cannot as yet be subjected to such an alyses because of difficulties in propagating HCV in tissue culture or in e xperimental animals. This enigma has been overcome by constructing a poliov irus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyse s to yield a virus (P/H710-d40) whose replication kinetics match that of th e parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby su pporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region w ithin domain II. Arguments that the phenotypes observed with the mutant chi mera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliov irus replicons with the gene order [PV] cloverleaf-[HCV]IRES-Delta core-R-L uc-[PV] IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IR ES-dependent translation independently from the replication of the replicon RNA.