Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278-303) and new roles for stem-loops H2 (310-352) and H3 (355-374)

Abstract

Genomic RNAs from retroviruses are packaged as dimers of two identical RNA molecules. In Moloney murine leukemia virus, a stem-loop structure (H1) located in the encapsidation domain Psi (nucleotides 215-564) was postulated to trigger RNA dimerization through base pairing between auto complementary sequences. The Psi domain also contains two other stem-loop structures (H2 and H3) that are essential for RNA packaging. Since it was suspected than H1 is not the only element involved in RNA dimerization, we systematically investigated the dimerization capacity of several subdomains of the first 725 nucleotides of genomic RNA. The efficiency of dimerization of the various RNAs was estimated by measuring their apparent dissociation constants, and the specificity was tested by competition experiments. Our results indicate that the specificity of dimerization of RNA nucleotides 1-725 is driven by motifs H1-H3 in domain Psi. To define the relative contributions of these elements, RNA deletion mutants containing different combinations of H1-H3 were constructed and further analyzed in competition and kinetic experiments. Our results confirm the importance of H1 in triggering dimerization and shed new light on the mechanism of dimerization. H1 is required to provide a stable dimer, probably through the formation of extended intermolecular interactions. However, H1-mediated association is a slow process that is kinetically enhanced by H3, and to a lesser extent by H2. We suggest that they facilitate the recognition between the two RNAs, most likely through their conserved GACG loops. Our results reinforce the idea that dimerization and packaging are two closely related processes.