Mutational robustness and resilience of a replicative cis-element of RNA virus: Promiscuity, limitations, relevance

Since replication of RNA-viruses is generally a low-fidelity process, it would be advantageous, if specific interactions of their genomic cis-elements with dedicated ligands are relatively tolerant to mutations. The specificity/promiscuity trade-off of such interactions was addressed here by investigating structural requirements of the oriL (also known as the clover leaf-like element), of poliovirus RNA, a replicative cis-element containing a conserved essential tetraloop functionally interacting with the viral protein 3CD. The sequence of this tetraloop and 2 adjacent base-pairs was randomized in the viral genome, and viable viruses were selected in susceptible cells. Strikingly, each position of this octanucleotide in 62 investigated viable viruses could be occupied by any nucleotide (with the exception of one position, which lacked U), though with certain sequence preferences, confirmed by engineering mutant viral genomes whose phenotypic properties were found to correlate with the strength of the cis-element/ligand interaction. The results were compatible with a hypothesis that functional recognition by 3CD requires that this tetraloop should stably or temporarily adopt a YNMG-like (Y=U/C, N=any nucleotide, M=A/C) fold. The fitness of "weak" viruses could be increased by compensatory mutations "improving" the tetraloops. Otherwise, the recognition of "bad" tetraloops might be facilitated by alterations in the 3CD protein. The virus appeared to tolerate mutations in its cis-element relaying on either robustness (spatial structure degeneracy) or resilience (a combination of dynamic RNA folding, low-fidelity replication modifying the cis-element or its ligand, and negative selection). These mechanisms (especially resilience involving metastable low-fit intermediates) can also contribute to the viral evolvability.