The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play essential roles at various stages of the viral life cycle. Through a novel assay we had recently developed, we reported on the necessary and sufficient region for RNA dimerization in the HIV-1 virion. Using this system, we performed further detailed mapping of the functional base pairs necessary for HIV-1 DLS structure. Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions. Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation. Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies.