Abstract
Five revertants of a linker-scanning mutation adjacent to the stem-loop V attenuation determinant (X472) in the 5′ noncoding region of poliovirus RNA were independently isolated from neuroblastoma cells and contained RNAs with seven nucleotide changes in the pyrimidine-rich region. Generation of the identical rare second-site mutations suggests the existence of a replicase-dependent mutagenesis mechanism during poliovirus replication. Enzymatic structure probing of the mutated pyrimidine-rich domain identified secondary structure changes between stem-loops V and VI. A consensus secondary structure model is presented for wild-type stem-loops V and VI and the pyrimidine-rich region located in the 5′ noncoding region of poliovirus RNA. A pyrimidine-rich region mutant (X472-R4N) produced large plaques in neuroblastoma cells and small plaques in HeLa cells, but the plaque size differences were not due to cell-type differences in viral translation or RNA replication. Release of X472-R4N from HeLa cells was 10-fold lower than release from neuroblastoma cells, which may explain the small plaque phenotype of X472-R4N in HeLa cells. Wild-type poliovirus was also released more efficiently from neuroblastoma cells (∼4-fold increase compared with release from HeLa cells), indicating that poliovirus neurotropism may be influenced by the cell-type efficiency of virus release. Thermal treatment increased the levels of infectious X472-R4N virions but not wild-type virus particles; thus RNA sequence and structural changes in the mutated 5′ noncoding region of X472-R4N may have altered RNA–protein interactions necessary for virus infectivity.
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