Identification of two functionally redundant RNA elements in the coding sequence of poliovirus using computer-generated design

Genomes of RNA viruses contain multiple functional RNA elements required for translation or RNA replication. We use unique approaches to identify functional RNA elements in the coding sequence of poliovirus (PV), a plus strand RNA virus. The general method is to recode large segments of the genome using synonymous codons, such that protein sequences, codon use, and codon pair bias are conserved but the nucleic acid sequence is changed. Such recoding does not affect the growth of PV unless it destroys the sequence/structure of a functional RNA element. Using genetic analyses and a method called "signal location search," we detected two unique functionally redundant RNA elements (α and β), each about 75 nt long and separated by 150 nt, in the 3'-terminal coding sequence of RNA polymerase, 3D(pol). The presence of wild type (WT) α or β was sufficient for the optimal growth of PV, but the alteration of both segments in the same virus yielded very low titers and tiny plaques. The nucleotide sequences and predicted RNA structures of α and β have no apparent resemblance to each other. In α, we narrowed down the functional domain to a 48-nt-long, highly conserved segment. The primary determinant of function in β is a stable and highly conserved hairpin. Reporter constructs showed that the α- and β-segments are required for RNA replication. Recoding offers a unique and effective method to search for unknown functional RNA elements in coding sequences of RNA viruses, particularly if the signals are redundant in function.