Pyrimidine-rich region mutations compensate for a stem-loop V lesion in the 5 ' noncoding region of poliovirus genomic RNA

Five revertants of a linker-scanning mutation adjacent to the stem-loop V a ttenuation determinant (X472) in the 5' noncoding region of poliovirus RNA were independently isolated from neuroblastoma cells and contained RNAs wit h 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 s tructure probing of the mutated pyrimidine-rich domain identified secondary structure changes between stem-loops V and VI. A consensus secondary struc ture model is presented for wild-type stem-loops V and VI and the pyrimidin e-rich region located in the 5' noncoding region of poliovirus RNA. A pyrim idine-rich region mutant (X472-R4N) produced large plaques in neuroblastoma cells and small plaques in HeLa cells, but the plaque size differences wer e 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 ne uroblastoma cells, which may explain the small plaque phenotype of X472-R4N in HeLa cells. Wild-type poliovirus was also released more efficiently fro m neuroblastoma cells (similar to 4-fold increase compared with release fro m 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; t hus RNA sequence and structural changes in the mutated 5' noncoding region of X472-R4N may have altered RNA-protein interactions necessary for virus i nfectivity. (C) 1999 Academic Press.