HIV-1 Maturation Inhibitor Bevirimat Stabilizes the Immature Gag Lattice

Abstract

Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVMtreated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly

“…The data presented here indicate that cleavage between CA and MA could even permit β-hairpin formation-and potentially rearrangement of the NTD-without destabilizing the immature CA-CTD arrangement. Lowresolution structural studies of inhibited viruses suggest that the bevirimat class of inhibitors, which inhibit proteolytic cleavage at the CA-SP1 boundary, stabilize an immature conformation of the lattice (39). We speculate that bevirimat could also arrest the virus in such a hybrid maturation state, in which partial maturation of the CA-NTD has already occurred.…”

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

“…Particles were prepared for cryo-ET as previously described (36). 293T or COS cells were transfected with HIV-1 molecular clone pNL4-3 (47), pNL4-3/PR Ϫ (48), or pNL4-3/CA5 (26).…”

“…Cryo-ET was performed as previously described (36,43). Inactivated purified virus was mixed with a solution of 10-nm colloidal gold particles (EMS) to serve as fiducial alignment markers during reconstruction.…”

“…Virus particles were extracted from raw and denoised reconstructions for subsequent analysis. Particles containing shell structures analyzed for lattice features were subject to subtomogram averaging, carried out as previously described (36). Briefly, shells of interest were modeled in three dimensions (3D) as a sphere of points, from which the area(s) of interest was selected and 3-dimensional subvolumes were extracted from around each point.…”

“…The spacer-called SP1 for HIV-1 and SP for RSV-plays an important role in assembly and maturation as attested by the observation that interventions affecting this region have drastic consequences (26,27,(30)(31)(32)(33)(34). Specifically, blockage of CA-SP1 cleavage by maturation inhibitors, such as the class prototype bevirimat (BVM), results in virions with severely reduced infectivity, accompanied by failure to assemble a mature core (2,(35)(36)(37).…”