Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

Abstract

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti–HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

Binding kinetics and substrate selectivity in HIV-1 protease−Gag interactions probed at atomic resolution by chemical exchange NMR

“…The Gag polyprotein comprises three ordered domains, matrix (MA), capsid (CA), and nucleocapsid (NC), connected by linkers and organized as follows: MA-CA-spacer peptide 1 (SP1)-NCspacer peptide 2 (SP2)-p6. The MAjCA linker, SP1, SP2, and p6 are intrinsically disordered in solution such that the ordered domains behave like beads on a string (4)(5)(6)(7).…”

“…As the Gag polyprotein is refractory to crystallization, all crystallographic work on PR−Gag interactions has been carried out using peptide analogs. Such peptides, however, are poor substitutes for the Gag polyprotein, since there are large differences in proteolysis rates of peptides and Gag under identical experimental conditions (6,15). The latter observations imply the presence of additional factors governing PR−Gag interactions, as well as a role for the ordered domains of Gag.…”

In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix

“…Based on NMR and X ray crystallography studies, p17 comprises five major alpha helices connected primarily by short loops (33,40). The C terminus of matrix is predicted to be disordered, which has hampered efforts to characterise the structural characteristics of this region.…”

“…Cleavage site mutations are thought to partially restore efficient cleavage by protease in the presence of bound drug (30,31). The mechanism for non-cleavage site mutations may include allosteric changes in protease-gag interactions that influence the efficiency by which protease locates cleavage sites through dynamic intermolecular interactions in the presence of drug (32,33). For example, our group previously reported the emergence of T81A in Gag that appeared to correlate with reduced susceptibility to the modern PI lopinavir in a subtype AG infected individual in France (26).…”

“…For example, our group previously reported the emergence of T81A in Gag that appeared to correlate with reduced susceptibility to the modern PI lopinavir in a subtype AG infected individual in France (26). This mutation was predicted to impact intermolecular interactions between Gag and protease by Deshmukh and colleagues using nuclear magnetic resonance (NMR) (33).…”