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

Antibodies inhibit human immunodeficiency virus type 1 (HIV-1) infection by targeting the envelope glycoprotein (Env). Env cleavage is a key determinant of antibody binding, as cleavage reduces Env flexibility and alters glycosylation. Given the greater abundance of uncleaved Env on the cell surface compared to virions, we investigated whether Env endocytosis, initiated through a membrane-proximal tyrosine motif in its cytoplasmic tail, regulates the abundance of cleaved Env at the cell surface. We hypothesize that such a shift would alter the cleavage and glycosylation profile of cell surface Env, affecting the sensitivity of cell-to-cell infection to neutralizing antibodies. To address this, we generated an endocytic mutant (ASPI-Env) and compared its antigenic properties to a cleavage-site mutant (SEKS-Env). Immunoprecipitation and ratiometric antibody binding studies of cell surface Env demonstrated that the ASPI mutation increases the amount of uncleaved Env on the cell surface. Consequently, ASPI-Env in cell-free infection was more sensitive to NAbs recognizing uncleaved Env. Notably, only b12, which can engage both cleaved and uncleaved Env but has a higher affinity for uncleaved Env, showed increased inhibition of ASPI-Env during cell-to-cell infection. This mirrors the enhanced neutralization of SEKS-Env during the cell-to-cell transfer assay and supports a model in which uncleaved Env participates in CD4-dependent virion transfer. Finally, the ASPI mutation altered lectin binding and differentially affected cell-to-cell and cell-free infections. Together, these findings indicate that Env endocytosis modulates the abundance of cleaved Env at the cell surface, thereby influencing antibody neutralization and lectin recognition in distinct modes of HIV-1 transmission. IMPORTANCEWe find that Env endocytosis modulates the antigenicity of Env on both the cell surface and virions. Blocking internalization increased uncleaved Env on the cell surface, thereby reshaping HIV-1 neutralization due to enhanced binding of antibodies that preferentially recognize uncleaved Env. We further show that uncleaved Env on the cell surface can initiate CD4-dependent transfer of virions across virological synapses. These findings demonstrate that Env endocytosis shapes cleavage-associated antigenicity in a manner that can differentially impact the recognition and neutralization of cells and viruses. The impact of uncleaved Env on neutralization indicates that a broader spectrum of cleaved and uncleaved Env conformations may be relevant when designing vaccines and cure strategies that must contend with a diverse antigenic landscape.