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.