Lenacapavir binding to immature Gag triggers the emergence of giant HIV-1 virions

Lenacapavir (LEN), a potent capsid inhibitor, suppresses reverse transcription and nuclear import by disrupting capsid core formation in human immunodeficiency virus type 1 (HIV-1). However, its effect on the late-stage viral assembly remains unclear. Although p24 ELISA suggested that LEN inhibited HIV-1, both RT-dPCR and Vpr-HiBiT assays indicated no reduction in the viral output. This discrepancy was attributed to LEN-induced epitope masking of p24, which was reversed by altered sample processing. Furthermore, LEN induced numerous abnormal, discrete clusters of Gag at the plasma membrane. Mass photometry and transmission electron microscopy revealed heterogeneous viral-like particles (LENiVLPs; diameter >200 nm), containing both Gag and Env proteins. Although LENiVLPs retained membrane fusion capability, they were non-infectious owing to post-entry defects. Thus, LEN binds to the precursor Gag and promotes aberrant particle formation, offering novel insights that could guide the development of next-generation LEN-based therapeutics targeting the late stages of the viral life cycle.