Restriction of HIV-1 by rhesus TRIM5α is governed by alpha helices in the Linker2 region.

TRIM5 alpha proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5 alpha proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5 alpha molecules within these assemblies have remained unclear. Here we show that alpha-helical elements within the Linker2 region of rhesus macaque TRIM5 alpha govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce alpha-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the alpha-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5 alpha dimer suggests a model in which alpha-helical elements within the Linker2 region dock to alpha-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5 alpha assembly and restriction and demonstrate that the antiviral potency of TRIM5 alpha proteins can be significantly increased without altering the affinity of SPRY/capsid binding.