Conformational activation and disulfide exchange in HIV-1 Env induce cell-free lytic/fusogenic transformation and enhance infection

Disulfide exchange is underexplored as a mechanism influencing HIV-1 entry. Prior studies demonstrated that redox enzyme inhibition can prevent HIV-1 infection but with limited mechanistic explanation. We hypothesize that ligand-driven rearrangement ("conformational activation") enables enzyme-mediated disulfide exchange in Env residues ("disulfide trigger") that promotes fusion transformations, enhancing virus entry. We tested soluble CD4 and CD4-binding site entry inhibitors as conformational activators and the ubiquitous redox enzyme thioredoxin-1 (Trx1) as disulfide trigger. We found that combination treatment caused fusion-like Env transformation and pseudovirus lysis, independent of cells. Notably, only compounds associated with gp120 shedding caused lysis when paired with Trx1. In each case, lysis was prevented by adding the fusion inhibitor T20, demonstrating that six-helix bundle formation is required as in virus-cell fusion. In contrast to conformationally activating ligands, neither the ground state stabilizer BMS-806 with Trx1 nor Trx1 alone caused lysis. Order of addition experiments reinforced conformational activation/disulfide trigger as a sequential process, with virus/activator preincubation transiently enhancing lysis and virus/Trx1 preincubation reducing lysis. Lastly, addition of exogenous Trx1 to typical pseudovirus infections exhibited dose-dependent enhancement of infection. Altogether, these data support conformational activation and disulfide triggering as a mechanism that can induce and enhance the fusogenic transformation of Env.IMPORTANCEHIV remains a global epidemic despite effective anti-retroviral therapies (ART) that suppress viral replication. Damage from early-stage infection and immune cell depletion lingers, as ART enables only partial immune system recovery, making prevention of initial virus entry preferable. In this study, we investigate disulfide exchange and its facilitating conformational rearrangements as underexplored, but critical, events in the HIV entry process. The HIV envelope (Env) protein effects cell entry by conformational rearrangement and pore formation upon interaction with immune cell surface proteins, but this transformation can be induced by Env's conformational activation and disulfide exchange by redox enzymes, which then integrates into established processes of HIV entry. The significance of this research is in identifying Env's conformational activation as a mechanistic requirement for initiating fusion by triggering disulfide exchange. This will aid the development of novel preventative strategies against HIV entry, particularly in the context of HIV-enhanced inflammation and comorbidities with redox mechanisms.