Replication of poliovirus RNA containing two VPg coding sequences leads to a specific deletion event

Studies of the poliovirus genome-linked protein VPg have shown that this small viral protein is required for replication of virus-specific RNA (Q. Reuer, R. J. Kuhn, and E. Wimmer, J. Virol. 64:2967-2975, 1990). To understand the mechanism of RNA replication, we constructed a recombinant poliovirus genome encoding two tandemly arranged VPg coding sequences that were nearly identical in both nucleotide and amino acid sequence. Following transfection of this two-VPg-containing RNA into HeLa cells, we found a specific and selective deletion in the progeny virus genome. Sequence analysis of the recovered viral RNA indicated that the complete nucleotide sequence encoding the second (3C-proximal) VPg coding sequences was removed, restoring the authentic genome sequences in the poliovirus genome. Analysis of viral RNAs following transfection suggested that the deletion event occurred during genome replication. Deletion could have occurred via homologous recombination between two VPg sequences or via intramolecular deletion with loop-out of the template. In vitro translation of the two-VPg-containing transcript RNA indicated aberrant processing of the viral polyprotein. This result suggested that selection of the wild-type genotype in the transfected cells may occur at the level of viral protein synthesis.