The primary mechanism for highly potent inhibition of HIV-1 maturation by lenacapavir.

Lenacapavir (LEN) is a highly potent, long-acting antiretroviral medication for treating people infected with muti-drug-resistant HIV-1 phenotypes. The inhibitor targets multifaceted functions of the viral capsid protein (CA) during HIV-1 replication. Previous studies have mainly focused on elucidating LEN's mode of action during viral ingress. Additionally, the inhibitor has been shown to interfere with mature capsid assembly during viral egress. However, the mechanism for how LEN affects HIV-1 maturation is unknown. Here, we show that pharmacologically relevant LEN concentrations do not impair proteolytic processing of Gag in virions. Instead, we have elucidated the primary mechanism for highly potent inhibition of HIV-1 maturation by sub-stoichiometric LEN:CA ratios. The inhibitor exerts opposing effects on formation of CA pentamers versus hexamers, the key capsomere intermediates in mature capsid assembly. LEN impairs formation of pentamers, whereas it induces assembly of hexameric lattices by imposing an opened CA conformation and stabilizing a dimeric form of CA. Consequently, LEN treatment results in morphologically atypical virus particles containing malformed, hyper-stable CA assemblies, which fail to infect target cells. Moreover, we have uncovered an inverse correlation between inhibitor potency and CA levels in cell culture assays, which accounts for LEN's ability to potently (with pM EC Author Summary: Lenacapavir (LEN) is the first-in-class HIV-1 capsid targeting antiretroviral that exhibits multimodal modality to inhibit both early and late steps of viral replication. Our studies here have elucidated previously undescribed structural and mechanistic bases for a highly potent antiviral activity of LEN during viral egress. These findings will inform clinical applications of LEN as a potent HIV-1 maturation inhibitor and aid the development of second-generation inhibitors targeting assembly of the mature viral capsid.