Poliovirus/hepatitis C virus (internal ribosomal entry site-core) chimericviruses: Improved growth properties through modification of a proteolytic cleavage site and requirement for core RNA sequences but not for core-related polypeptides

H.-H, Lu and E. Wimmer (Proc. Natl, Acad, Sci. USA 93:1412-1417, 1996) have demonstrated that the internal ribosomal entry site (IRES) of poliovirus ( PV) can be functionally replaced by the related genetic element from hepati tis C virus (HCV), One important finding of this study was that open readin g frame sequences 3' of the initiating AUG, corresponding to the open readi ng frame of the HCV core polypeptide, are required to create a viable chime ric virus. This made necessary the inclusion of a PV 3C protease (3C(pro)) cleavage site for proper polyprotein processing to create the authentic N t erminus of the PV capsid precursor. Chimeric PV/HCV (P/H) viruses, however, grew poorly relative to PV, The goal of this study was to determine the mo lecular basis of impaired replication and enhance the growth properties of this chimeric virus. Genetic modifications leading to a different proteinas e (PV 2A(pro)) cleavage site between the HCV core sequence and the PV polyp rotein (P/H701-2A) proved far superior with respect to viral protein expres sion, core-PV fusion polyprotein processing, plaque phenotype, and viral ti ter than the original prototype PV/HCV chimera containing the PV 3C(pro)-sp ecific cleavage site (P/H701). We have used this new virus model to answer two questions concerning the role of the HCV core protein in P/H chimeric v iral proliferation. First, a derivative of P/H701-2A with frameshifts in th e core-encoding sequence was used to demonstrate that production of the cor e protein was not necessary for the translation and replication of the P/H chimera. Second, a viral construct with a C-terminal truncation of 23 amino acids of the core gene was used to show that a signal sequence for signal peptidase processing, when present in the viral construct, is detrimental t o P/H virus growth. The novel P/H chimera described here are suitable model s for analyzing the function(s) of the HCV elements by genetic analyses in vivo and for antiviral drug discovery.