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'-n on-coding region (NCR) reduce neurovirulence and cell-specific cap-independ ent translation. The stem-loop V attenuation determinants lie within the hi ghly structured internal ribosome entry site, Although stem-loop V Sabin mu tations have been proposed to alter RNA secondary structure, efforts to ide ntify such conformational changes have been unsuccessful, A previously desc ribed linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transf ection of X472 RNA generated only pseudo-revertants in HeLa (cervical carci noma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell ty pes contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located similar to 100 nt distal to the original le sion. Enzymatic RNA structure probing determined that the X472 linker subst itution did not disrupt the overall conformation of stem-loop V but abolish ed base pairing adjacent to the attenuation determinant. Our analyses corre lated increased base pairing proximal to the stem-loop V attenuation determ inant with growth of X472 revertant RNAs (measured by northern blot analysi s). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are sho wn on a consensus secondary structure model for stem-loop V of the PV 5'-NC R.