POLIOVIRUS RNA RECOMBINATION IN CELL-FREE-EXTRACTS

Poliovirus RNA has been shown to undergo homologous genetic recombinat ion at a high frequency in infected human cells. Recently it has becom e possible to mimic the entire intracellular replicative cycle of poli ovirus replication in cytoplasmic extracts prepared from HeLa cells, r esulting in the generation of infectious poliovirions. The mechanism o f poliovirus RNA recombination has been shown previously to be coupled to RNA replication, presumably by template switching during the repli cation of parental RNAs. Experiments were designed to test whether rec ombinant poliovirus RNA molecules are produced in a cell-free environm ent. Recombinant molecules generated bear marker sequences that can be detected physically by reverse transcription and PCR. We report here successful detection of poliovirus RNA recombination in a cell-free re plication system. The frequency measured for cell-free RNA recombinati on between two polymorphic marker loci 656 nt apart was between 10(-2) and 10(-3) recombinants/genome, a frequency comparable to or slightly higher than that measured for RNA recombination in infected cells.