Abstract Body

Background: Entry of HIV-1 into host cells remains a compelling target for developing agents to prevent infection. This step is mediated by a sequence of interactions of a trimeric gp120/gp41 virus envelope (Env) protein complex with host cells. Peptide triazoles, a class of entry inhibitors have shown to bind to gp120 with close to nanomolar affinity, to suppress protein ligand interactions of gp120 with CD4 and co-receptor binding sites, and to inhibit cell infection by a broad range of virus subtypes. We found that sulfhydryl-containing peptide triazoles (PT-SH), irreversibly disrupts cell-free virions, leading to virus core release. We seek to understand the mechanism of this cell-free virolysis. We hypothesize that the PT-SH hijacks the viral fusion mechanism prior to cell fusion and leads to transient pore formation resulting in irreversible virolysis. Methodology: The peptide triazoles (PTs) were synthesized using solid phase Fmoc chemistry with click conjugation. The PTs included KR13 (PT-SH); KR13b (KR13 with the thiol blocked); KR13s (containing a scrambled KR13 sequence), and the parent PT, HNG156, with no free thiol. Infection inhibition by PTs were compared using a single-round infection assay using lab synthesized pseudoviruses. Release of Env gp120 and nucleocapsid protein p24 from the PT treated virions were tested by western blot and ELISA respectively. The fixed residual virion was tested for gp41 recognition by direct ELISA using antibodies 2F5 and 4E10 and morphological analysis using transmission electron microscopy. To compare the relationship of KR13 induced cell-free virolysis and virus-cell entry, we examined the effect of T20 on the inhibition of KR13 induced virolysis. Results: The PTs with active pharmacophore (KR13, HNG156 and KR13b) all inhibit viral infection and induce gp120 shedding; uniquely only KR13 induced release of the core protein p24. Viral inactivation and gp120 shedding had a similar time-dependence, while p24 release lagged temporally. The resulting virions lacked an organized capsid and the gp41 on the virions were antigenically active against neutralizing antibodies, 2F5 and 4E10. Viral lysis was completely inhibited by T20, which blocks formation of the gp41 6-helix bundle during membrane fusion. Conclusions: Our data are consistent with a model in which PT-SH triggers physiologic structural changes in the HIV-1 Env including the 6 helix bundle formation that is typically associated with viral fusion and entry. The potency and specific activity of this novel compound and its ability to inactivate virions prior to target cell engagement suggest that KR13 could be highly effective as a microbicide in HIV prevention and transmission as well as a probe to understand biochemical signals required for virus-cell fusion