The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein

The major packaging signal of human immunodeficiency virus type 1 (HIV-1) R NA has been localised to the region 3' to the major splice donor within the leader sequence. Secondary structural studies for this region of the HIV-1 genome have shown the existence of a stem-loop structure capped by a purin e-rich tetraloop. Extensive mapping data presented here lead to the complet e characterisation of the structure of the stem-loop, including a new purin e-rich internal loop in the lower part of the structure and the previously established GGAG tetraloop at its tip. Biochemical analysis reveals that bo th internal loop and tetraloop are primary sites for interaction with Gag p olyprotein, and that binding of Gag protein leads to a conformational chang e which alters the RNA structure. NMR spectroscopy has been used to determi ne the three-dimensional structure of this complete stem-loop structure. Th e structural analysis reveals a significant difference between the apical p art of the stem-loop structure, which adopts a well-defined conformation, a nd the purine-rich internal loop, which is instead very flexible. In contra st to what is generally observed for internal loop structures in RNA, this region of the encapsidation signal adopts a structure lacking stable inters trand interactions capable of stabilising a unique conformation. We suggest that the stem-loop structure represents a nucleation site for Gag protein binding, and that the protein exploits the flexibility of the internal loop to initiate the unwinding of the structure with successive addition of Gag molecules interacting with the RNA and each other through conserved I (int eraction) domains. (C) 2000 Academic Press.