Mutational analysis of the 3'-terminal extra-cistronic region of poliovirus RNA: secondary structure is not the only requirement for minus strand RNA replication.
A series of mutations were introduced in the 3'-terminal untranslated region (3'-UTR) of full-length infectious poliovirus cDNA clones, and following transfection of COS-1 cells the ability of these constructs to generate viable viral particles and/or to support viral RNA synthesis was assayed. Substitution of the 3'-UTR of poliovirus RNA with the equivalent sequences of HAV RNA abrogated viral RNA replication, whereas the introduction of extended 'foreign' sequences between the open reading frame and the 3'-UTR was well tolerated. Point mutation that either destabilized the stem-and-loop structure or altered the sequence of the loop in domain 'Y' (nomenclature as per Pilipenko et al., [Nuclei Acids Res. 20 (1992) 1739-1745]) abolished both the infectivity and viral RNA synthesis. These were not restored by compensatory mutation that reconstructed the native secondary structure of this domain, suggesting that the secondary/tertiary folding of the 3'-UTR is not the only determinant for template recognition at initiation of RNA synthesis, but rather that a specific primary sequence is indeed required.
Pierangeli
A
A
Department of Cellular and Developmental Biology, University of Rome, Italy.
Bucci
M
M
Pagnotti
P
P
Degener
A M
AM
Perez Bercoff
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005796:Genes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D017955:Poliovirus / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
D012367:RNA, Viral; C060630:endodeoxyribonuclease NaeI; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific