Abstract

A betulinic acid-based compound, bevirimat (BVM), the first-in-class HIV-1 maturation inhibitor (MI), acts by blocking a late step of Gag processing, capsid-spacer peptide 1 (CA-SP1) cleavage. PF-46396 (PF96) was found to have a similar mode of action despite being structurally distinct from BVM. Although in clinical trials BVM reduced viral loads in HIV-1-infected patients, single-amino-acid polymorphisms in the SP1 region resulted in reduced susceptibility to the compound. To overcome this problem, we developed “second-generation” MIs based on the BVM scaffold. We identified a set of BVM analogs that show potent and broad antiviral activity.

To understand the target and mechanism of action of MIs, we selected for viral resistance. BVM resistance mapped to residues surrounding the CA-SP1 cleavage site, whereas PF96-resistance mutations clustered at the cleavage site and far upstream in CA, including within the major homology region (MHR). Interestingly, a group of these MHR mutants were profoundly PF96-dependent. Propagation of the MHR mutants led to the selection of a second-site compensatory change at SP1 residue 8 (SP1-T8I). Moreover, on its own, the SP1-T8I mutation induced a high level of CA-SP1 accumulation. The SP1-T8I mutation also stabilized the immature Gag lattice. These results indicate that SP1-T8I phenocopies the effect of MI binding.

Selection experiments with second-generation BVM analogs identified the mutation CA-P157A, located in the MHR. Pulse-chase data demonstrate that CA-SP1 processing kinetics for CA-P157A are similar to those of the WT. The characterization of resistant mutants provides novel insights into the structure of the MI-binding site and the role of SP1 and the CA MHR in virus assembly and viral maturation.