Poly(rC) binding proteins mediate poliovirus mRNA stability

The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structur e and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBP s; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB B lyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the func tional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively ass ay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus n egative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild- type poliovirus mRNA unstable In these reactions. A 5'-terminal 7-methylgua nosine cap prevented the degradation of wild-type poliovirus mRNA In the pr esence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did n ot adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor R NA inhibited the translation of poliovirus mRNA but did not inhibit poliovi rus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' clover leaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguano sine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A m utation did not inhibit the translation of poliovirus mRNA nor diminish vir al negative-strand RNA synthesis relative to wild-type RNA. These data supp ort the conclusion that poly(rC) binding protein(s) mediate the stability o f poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of pol iovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequ ences among picornaviruses, including C24 in loop b of the cloverleaf, we s uggest that viral mRNA stability of polioviruses, coxsackieviruses, echovir uses, and rhinoviruses is mediated by interactions between PCBPs and 5' clo verleaf RNA.