The 5′-terminal 88 nt of poliovirus RNA fold into a
cloverleaf RNA structure and form ribonucleoprotein complexes
with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino,
RNA, 1997, 3:882–892; TB Parsley, JS
Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997,
3:1124–1134). To determine the functional role
of these ribonucleoprotein complexes in poliovirus replication,
HeLa S10 translation-replication reactions were used to
quantitatively assay poliovirus mRNA stability, poliovirus mRNA
translation, and poliovirus negative-strand RNA synthesis.
Ribohomopoly(C) RNA competitor rendered wild-type poliovirus
mRNA unstable in these reactions. A 5′-terminal
7-methylguanosine cap prevented the degradation of wild-type
poliovirus mRNA in the presence of ribohomopoly(C) competitor.
Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus
mRNA stability. Ribohomopoly(C) competitor RNA inhibited the
translation of poliovirus mRNA but did not inhibit poliovirus
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′ cloverleaf RNA and rendered
poliovirus mRNA unstable. A 5′-terminal 7-methylguanosine
cap blocked the degradation of C24A mutant poliovirus mRNA.
The C24A mutation did not inhibit the translation of poliovirus
mRNA nor diminish viral negative-strand RNA synthesis relative
to wild-type RNA. These data support the conclusion that poly(rC)
binding protein(s) mediate the stability of poliovirus mRNA
by binding to the 5′-terminal cloverleaf structure of
poliovirus mRNA. Because of the general conservation of 5′
cloverleaf RNA sequences among picornaviruses, including C24
in loop b of the cloverleaf, we suggest that viral mRNA stability
of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses
is mediated by interactions between PCBPs and 5′ cloverleaf
RNA.