RNA structure adjacent to the attenuation determinant in the 5'-non-coding region influences poliovirus viability.
In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.
Stewart
S R
SR
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697, USA.
Semler
B L
BL
D020121:5' Untranslated Regions / Q000502:physiology*; D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics / Q000821:virology; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D017955:Poliovirus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D011742:Pyrimidine Nucleotides / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000502:physiology*; D017423:Sequence Analysis, RNA; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000235:genetics
D020121:5' Untranslated Regions; D011742:Pyrimidine Nucleotides; D012367:RNA, Viral