REPLICATION OF POLIOVIRUS RNA CONTAINING 2 VPG CODING SEQUENCES LEADSTO A SPECIFIC DELETION EVENT

Studies of the poliovirus genome-linked protein VPg have shown that th is small viral Protein is required for replication of virus-specific R NA (Q. Reuer, R. J. Kuhn, and E. Wimmer, J. Virol. 64:2967-2975, 1990) . To understand the mechanism of RNA replication, we constructed a rec ombinant poliovirus genome encoding two tandemly arranged VPg coding s equences that were nearly identical in both nucleotide and amino acid sequence. Following transfection of this two-VPg-containing RNA into H eLa cells, we found a specific and selective deletion in the progeny v irus genome. Sequence analysis of the recovered viral RNA indicated th at the complete nucleotide sequence encoding the second (3C-proximal) VPg coding sequences was removed, restoring the authentic genome seque nces in the poliovirus genome. Analysis of viral RNAs following transf ection suggested that the deletion event occurred during genome replic ation. Deletion could have occurred via homologous recombination betwe en two VPg sequences or via intramolecular deletion with loop-out of t he template. In vitro translation of the two-VPg-containing transcript RNA indicated aberrant processing of the viral polyprotein. This resu lt suggested that selection of the wild-type genotype in the transfect ed cells may occur at the level of viral protein synthesis.