Endocytic motif on HIV-1 Env regulates cleavage status and antibody neutralization of cell-free and cell-to-cell infection.

Endocytic recycling regulates the abundance of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on the cell surface, where Env comprises both cleaved and uncleaved forms. In contrast, released virions are enriched for cleaved Env. Because cell-free infection depends on virion-associated Env, and cell-to-cell infection is initiated by cell-surface Env, we examined whether Env endocytosis alters the balance of cleaved and uncleaved Env available for antibody recognition and thereby influences neutralization sensitivity in each transmission mode. To address this, we engineered an endocytic-deficient (ASPI-Env) and a cleavage-deficient (SEKS-Env) Env in a transmitted/founder clade B background. We compared them with wild-type Env using surface immunoprecipitation, ratiometric antibody binding, and neutralization assays. Disruption of the endocytic motif increased surface exposure of uncleaved Env and shifted the antigenic profile toward an uncleaved Env phenotype. Infection by cell-free virus particles carrying ASPI-Env showed increased sensitivity to antibodies that preferentially recognize uncleaved or open Env conformations (b12 and 17b), while its sensitivity to the cleaved Env-preferring epitope recognized by PGT151 was similar to wild type. In contrast, cell-to-cell infection showed minimal changes in neutralization sensitivity across the antibody panel, except for b12, which exhibited increased potency and greater inhibition of cell-to-cell HIV-1 transfer. Additionally, uncleaved SEKS-Env functioned in a CD4-dependent virion transfer assay, indicating a previously unappreciated ability to initiate cell-to-cell viral transfer. Together, these findings suggest that Env endocytosis shapes the heterogeneous pool of Env engaged in infection, thereby influencing antibody engagement, with important implications for HIV-1 vaccine strategies. IMPORTANCE: Env endocytosis shapes the antigenic landscape of HIV-1 by influencing the cleavage state of Env on both cells and virions. Env lacking a dominant internalization signal displays more uncleaved Env at the cell surface, thereby enhancing recognition by antibodies that preferentially target open or uncleaved conformations. Notably, cleavage-deficient Env can support CD4-dependent, cell-to-cell viral transfer, indicating that uncleaved cell surface Env can participate in early events of cell‑to‑cell infection. Together, these results suggest that Env sorting pathways influence cleavage-associated antigenicity and underscore the importance of considering both cleaved and uncleaved Env conformations in vaccine and cure strategies.