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 picomolar EC₅₀ values) inhibit HIV-1 maturation at clinically relevant drug concentrations. Author summary: Lenacapavir (LEN) is used to treat adults with multi-drug-resistant HIV-1 infection. LEN is the first in class, highly potent and long-acting antiretroviral, which works by a unique mechanism of targeting the viral capsid protein. LEN is administered through subcutaneous injections twice-yearly in combination with other HIV-1 medications. The long-acting antiviral activity relies on the ability of sub-nanomolar LEN concentrations to inhibit HIV-1 replication. Accordingly, cell culture-based mechanistic studies are expected to focus on elucidating highly potent antiviral modes of action of LEN. We and others have previously reported the primary mechanism of inhibition of HIV-1 ingrees by LEN. Our studies here have deciphered previously undescribed mechanistic and structural bases for a highly potent antiviral activity of LEN during viral egress. Specifically, we show that clinically relevant LEN concentrations induce aberrant capsid protein assembly during virus maturation and consequently yield non-infectious HIV-1 paricles. 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. [ABSTRACT FROM AUTHOR]