The packaging signal of HIV-1 RNA contains a stem-loop structure, SL1, which serves as the dimerization initiation site for two identical copies of the genome and is important for packaging of the RNA genome into the budding virion and for overall infectivity. SL1 spontaneously dimerizes via a palindromic hexanucleotide sequence in its apical loop, forming a metastable kissing dimer form. Incubation with nucleocapsid protein causes this form to refold to a thermodynamically stable mature linear dimer. Here, we present an NMR structure of the latter form of the full-length SL1 sequence of the Lai HIV-1 isolate. The structure was refined using nuclear Overhauser effect and residual dipolar coupling data. The structure presents a symmetric homodimer of two RNA strands of 35 nucleotides each; it includes five stems separated by four internal loops. The central palindromic stem is surrounded by two symmetric adenine-rich 1-2 internal loops, A-bulges. All three adenines in each A-bulge are stacked inside the helix, consistent with the solution structures of shorter SL1 constructs determined previously. The outer 4-base pair stems and, proximal to them, purine-rich 1-3 internal loops, or G-bulges, are the least stable parts of the molecule. The G-bulges display high conformational variability in the refined ensemble of structures, despite the availability of many structural restraints for this region. Nevertheless, most conformations share a similar structural motif: a guanine and an adenine from opposite strands form a GA mismatch stacked on the top of the neighboring stem. The two remaining guanines are exposed, one in the minor groove and another in the major groove side of the helix, consistent with secondary structure probing data for SL1. These guanines may be recognized by the nucleocapsid protein, which binds tightly to the G-bulge in vitro.As with all retroviruses, two identical copies of genomic RNA are packaged in the viral particles of HIV-1.2 The non-covalent dimer formed by these two RNA strands is involved in several stages of the viral life cycle, such as reverse transcription, recombination, RNA packaging; its integrity is important for optimal viral infectivity (reviewed in Ref. 1). The packaging signal is located in the 5Ј-untranslated region of unspliced RNA; it contains four stem-loop structures, SL1, SL2, SL3, and SL4 (Fig. 1A). Three of them, SL1, SL2, and SL3 are specifically recognized in vitro by the NC domain of the viral Gag polyprotein (2), a key component of RNA packaging. SL1 is a 35-nt hairpin; its apical loop contains a 6-nt self-complementary sequence and three flanking residues, most commonly purines. This hairpin was identified as a dimerization initiation sequence (3, 4); RNA constructs containing SL1 are capable of spontaneously dimerizing in vitro via loop-loop interaction, forming metastable kissing dimer structures. Incubation of kissing dimers at 55°C converts them into a more stable linear, or mature, dimeric form with 1-2 internal loops, or A-bulges, being formed on both flan...