Convergence of natural and artificial evolution on an RNA loop-loop interaction: The HIV-1 dimerization initiation site

Loop-loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop-loop interaction at the d imerization initiation site (DIS), a hairpin located in the 5' noncoding re gion that contains an autocomplementary sequence in the loop. Only two majo r DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop-loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization- competent sequences. Surprisingly, increasing interloop complementarity abo ve a threshold of 6 bp did not enhance dimerization, although the combinati ons of nucleotides forming the theoretically most stable hexanucleotide dup lexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs dosing the loop and co variations between flanking and central loop nucleotides. Degeneration of t he entire loop yielded a complex population of dimerization-competent seque nces whose consensus sequence resembles that of wild-type HIV-1, We conclud e from these findings that the DIS has evolved to satisfy simultaneous cons traints for optimal dimerization affinity and the capacity for homodimeriza tion. Furthermore, the most constrained features of the DIS identified by o ur experiments could be the basis for the rational design of DIS-targeted a ntiviral compounds.