The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

Abstract

BackgroundBevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1.ResultsIn this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly.ConclusionsThis is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

“…We considered the possibility that either the SP sequence may form a direct intersubunit bridge to help promote protein assembly or, alternatively, the peptide may make an intrasubunit interaction with the CTD that influences allosterically the affinity of the CTD interactions. Either scenario would be consistent with observations of HIV suggesting that in the Gag lattice, the SP1 sequence lies in close proximity to the CTD, specifically the major homology region (MHR) (21,60,61). However, the relative insensitivity of CA-SP assembly to alanine or proline substitutions in the spacer peptide seems to argue against such scenarios that require a direct docking of SP against the CTD.…”

A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition

“…7A). We infer that the inhibitors impede displacive transition of the lattice, presumably by binding directly to it, as has been suggested elsewhere (3,56), and that BVM is more efficient in this regard. With PF-46396, the clamp-like effect of inhibitor binding is less stringent and displacive transition can initiate at one or more sites on the immature-like lattice and propagate outwards in a wave-like manner, creating the thin-walled patches that we observe.…”

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

“…Understanding the conformational transition of SP1 from an unstructured motif in monomeric/dimeric ΔGag to a helical conformation in an assembled Gag lattice, if this does indeed occur, will require atomic resolution structures of immature Gag assemblies. It is interesting to note that a six helix bundle of SP1 has been proposed as the binding site for a new class of anti-HIV drugs (Berivimat and related compounds) known as maturation inhibitors (38). No indication for any interaction between Berivimat (0.3 mM) and ΔGag (protein:drug molar ratio of 1:6) was observed by NMR under conditions where ΔGag exists in a monomer/dimer equilibrium (SI Materials and Methods).…”