Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

Significance The late stages of the HIV-1 life cycle are governed by the Gag polyprotein (also known as group specific antigen). Here we use modern solution NMR methods to probe the structure and conformation of Gag. We show that Gag exists in a dynamic monomer–dimer equilibrium in solution and its structured domains reorient semi-independently of one another. We monitor proteolytic processing of Gag by HIV-1 protease in real-time, and show that the conformational transition at the N-terminus of the capsid domain from a random coil to a β-hairpin upon cleavage at the matrix|capsid junction occurs five times faster than cleavage at the capsid|spacer peptide 1 junction. These results provide insights into the HIV-1 life cycle that may lead to the development new antiretroviral strategies. Assembly and maturation of the human immunodeficiency virus type 1 (HIV-1) are governed by the Gag polyprotein. Here we study the conformation and dynamics of a large HIV-1 Gag fragment comprising the matrix, capsid, spacer peptide 1 and nucleocapsid domains (referred to as ΔGag) by heteronuclear multidimensional NMR spectroscopy. In solution, ΔGag exists in a dynamic equilibrium between monomeric and dimeric states. In the presence of nucleic acids and at low ionic strength ΔGag assembles into immature virus-like particles. The structured domains of ΔGag (matrix, the N- and C-terminal domains of capsid, and the N- and C-terminal zinc knuckles of nucleocapsid) retain their fold and reorient semi-independently of one another; the linkers connecting the structural domains, including spacer peptide 1 that connects capsid to nucleocapsid, are intrinsically disordered. Structural changes in ΔGag upon proteolytic processing by HIV-1 protease, monitored by NMR in real-time, demonstrate that the conformational transition of the N-terminal 13 residues of capsid from an intrinsically disordered coil to a β-hairpin upon cleavage at the matrix|capsid junction occurs five times faster than cleavage at the capsid|spacer peptide 1 junction. Finally, nucleic acids interact with both nucleocapsid and matrix domains, and proteolytic processing at the spacer peptide 1|nucleocapsid junction by HIV-1 protease is accelerated in the presence of single-stranded DNA.