Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA–SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA–SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix–coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions.

Significance: The mechanisms of how TRIM5α interferes with the integrity of the HIV-1 capsid to restrict HIV-1 infectivity remain poorly understood. We examined, at atomic resolution, the interactions with TRIM5α in the assembled capsid and the dynamics of capsid’s hexameric and pentameric building blocks. Remarkably, assemblies in the presence of the pentameric subunits are more rigid on microsecond to millisecond timescales at the sites of pentamer incorporation than tubes containing only hexamers. Furthermore, TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces, essential for higher-order capsid assembly. TRIM5α thus uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our results suggest that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.