Lenacapavir binding to immature Gag induces giant virions and causes protease-dependent inhibition of viral release.

Lenacapavir (LEN), a potent capsid inhibitor, suppresses reverse transcription and nuclear import by disrupting capsid core formation in HIV type 1 (HIV-1). However, its effects on late-stage viral assembly remain unclear. Although p24 ELISA suggested that LEN substantially inhibited HIV-1, both RT-dPCR and Vpr-HiBiT assays indicated that suppression of viral release required relatively high LEN concentrations. This discrepancy was attributed to LEN-induced reduction in p24 solubility, leading to an overestimation of its inhibitory effect on viral release. In addition, excessive intracellular Gag processing by the viral protease was observed in the presence of LEN, which contributed to impaired viral release. Furthermore, LEN induced numerous abnormal, discrete clusters of Gag at the plasma membrane. Using mass photometry, which was applied to analyze HIV-1 particle sizes, and transmission electron microscopy, we identified heterogeneous LEN-induced viral-like particles (LENiVLPs; diameter >200 nm) containing both Gag and Env proteins. Although LENiVLPs retained membrane fusion capability, they were noninfectious owing to postentry defects that prevented viral replication. Thus, LEN binds to the precursor Gag and promotes aberrant particle formation, offering insights that could guide the development of next-generation LEN-based therapeutics targeting the late stages of the viral life cycle.