The 5′-untranslated region (5′-UTR) of the human immunodeficiency virus type-1 (HIV-1) genome regulates multiple RNA-dependent functions during viral replication and has been proposed to adopt multiple secondary structures. Recent phylogenetic studies identified base pair complementarity between residues of the unique 5′ element and those near the gag start codon (gagAUG) that is conserved among evolutionarily distant retroviruses, suggesting a potential long-range RNA-RNA interaction. However, nucleotide accessibility studies led to conflicting conclusions about the presence of such interactions in virions and in infected cells. Here we show that an 11-nucleotide oligo-RNA spanning residues 105-115 of the 5′-UTR (U5) readily binds to oligoribonucleotides containing the gag start codon (AUG), disrupting a pre-existing stem loop and forming a heteroduplex stabilized by 11 Watson Crick base pairs (Kd = 0.47 ± 0.16 μM). Addition of the HIV-1 nucleocapsid protein (NC), the transacting viral factor required for genome packaging, disrupts the heteroduplex by binding tightly to U5 (Kd = 122 ± 10 nM). The structure of the NC:U5 complex, determined by NMR, exhibits features similar to those observed in NC complexes with HIV-1 stem loop RNAs, including the insertion of guanosine nucleobases to hydrophobic clefts on the surface of the zinc fingers and a 3′-to-5′ orientation of the RNA relative to protein. Our findings indicate that the previously proposed long-range U5-gagAUG interaction is feasible, and suggest a potential NC-dependent mechanism for modulating the structure of the 5′-UTR.

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