Mechanistic Insights into Lenacapavir-Induced Off-Pathway HIV-1 Capsid Assembly

The HIV-1 capsid is a fullerene-like shell composed of hexamer and pentamer arrangements of the capsid (CA) proteins. The cone shape of the capsid is particularly important for packaging the viral genome and coordinating nuclear entry. Lenacapavir (LEN), a potent long-acting inhibitor, has been shown to disrupt capsid morphogenesis by binding at the FG-binding pocket located between neighboring CA subunits. Interestingly, inositol hexakisphosphate (IP6), a cellular polyanion, binds within the central pore of capsid pentamers and some hexamers while playing a key role in regulating the hexamer/pentamer switch. As LEN and IP6 interact with overlapping structural elements, they can compete to influence the capsid assembly pathway and outcomes. Using coarse-grained molecular simulations, we examined capsid assembly across varying IP6 and LEN conditions. Our results reveal a concentration-dependent shift in assembly outcomes: LEN accelerates hexamer assembly and reduces pentamer incorporation, leading to malformed, multilayered, or incomplete capsids. Simulations including a model for the viral ribonucleoprotein (RNP) complex further show that LEN-treated capsids frequently fail to encapsidate the RNA genome, indicating impaired maturation. Our calculations confirm that LEN impairs the formation of high-curvature CA lattice regions necessary for closure, supporting a model of off-pathway assembly as a mechanism of viral inhibition.