title: Poly(rC) binding proteins mediate poliovirus mRNA stability.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Colorado Health Sciences Center, Denver 80262, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Murray
authors.forename: A W
authors.initials: AW
authors.lastname: Roberts
authors.forename: D J
authors.initials: DJ
authors.lastname: Barton
mesh_terms: D001667:Binding, Competitive; D018076:DNA, Complementary / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics / Q000502:physiology*; D011061:Poly A; D011066:Poly C; D011068:Poly G; D011072:Poly U; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry*; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012367:RNA, Viral; D011061:Poly A; D011068:Poly G; D011072:Poly U; D011066:Poly C