Background:  Membrane cholesterol plays an important role in HIV-1 assembly, release, and infectivity. Here we investigate the target and mechanism of action of the cholesterol-binding compound amphotericin B methyl ester (AME), a water-soluble, relatively non-toxic derivative of the polyene fungal antibiotic amphotericin B.

Methods:  To investigate the potential anti-HIV-1 activity of AME we studied the effect of AME on HIV-1 replication. To determine the effect of AME on virus infectivity, we performed single-cycle assays in HeLa-derived TZM cells. We also performed virus release assays to study the effect of AME on virus assembly and release. Finally, to understand the mechanism of action of AME, we selected for, isolated, and characterized AME-resistant HIV-1 variants.

Results:  We observed that AME blocks the replication of diverse HIV-1 isolates, irrespective of their clade, target cell tropism, or resistance to RT or PR inhibitors. We show that AME profoundly impairs virus entry and also induces a ~5-fold decrease in virus particle production. The mutations responsible for AME resistance, P203L and S205L, mapped to an endocytosis motif in the cytoplasmic tail of gp41. Virus replication and single-cycle infectivity assays confirmed that the P203L and S205L substitutions confer AME resistance. Interestingly, truncation of the gp41 cytoplasmic tail of either HIV-1 or SIV_{mac gp41} also renders these primate lentiviruses resistant to AME. We investigated the connection between point mutations in the gp41 cytoplasmic tail and gp41 truncation. Remarkably, we observed that the AME-resistance mutations induce the cleavage of the gp41 cytoplasmic tail by the viral PR.

Conclusions:  In response to inhibition by AME, HIV-1 has evolved a strategy used by murine leukemia virus and Mason-Pfizer monkey virus to activate Env fusogenicity. Like these distantly related retroviruses, AME-resistant HIV-1 variants retain the Env cytoplasmic tail until Env has been incorporated, then cleave off the tail with PR to activate fusogenicity. This study identifies a novel mechanism of resistance to an HIV-1 entry inhibitor and represents the first instance in which HIV-1 Env function is activated by PR-mediated Env cleavage.