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

Abstract

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, 3Dpol . 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.computer-designed sequences | chemical DNA synthesis

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

“…Some of the alanine substitutions in our study (S416A, L419A, and L420A) involved changes in the sequence of an RNA element within the 3D gene (42), but the RNA sequences within the 3D gene are unlikely to affect the frequency of replicative RNA recombination. Two of these alanine substitutions (S416A and L419A) exhibited increased RNA recombination, whereas the third (L420A) exhibited dramatically reduced RNA recombination.…”

“…Two of these alanine substitutions (S416A and L419A) exhibited increased RNA recombination, whereas the third (L420A) exhibited dramatically reduced RNA recombination. The role of 3D pol in replicative RNA recombination is generally accepted (21,25), while the functions of RNA elements in the 3D gene remain ambiguous (42,43). Consequently, we attribute phenotypic changes in the frequency of RNA recombination to the alanine substitutions in 3D pol rather than to changes in RNA sequences encoding 3D…”

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

“…This describes the frequency of synonymous codon pairs that does not fit the use of individual codons (16)(17)(18) (1). The loci of three replication-essential hairpin structures, Cre (81), and α and β (23,24), as well as the conserved but not-essential RNase L ci element (25,26), are shown above the PP. The SnaB I and Bgl II restriction sites, used for subcloning the P2 and P3 domains, are indicated.…”

Diverse roles and interactions of RNA structures during the replication of positive-stranded RNA viruses of humans and animals

“…However, the dynamics of their interactions within host cells and the role of other regions of structured RNA remain to be fully characterized. For example, two recent studies revealed a number of novel structures within the ORF (Burrill et al, 2013;Song et al, 2012), one of which -element 3D-7000 -is implicated in RNA replication through an as yet unknown mechanism (Burrill et al, 2013).…”