Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Significance One of the most notable features of TRIM5 proteins is their ability to restrict retroviral infections by binding viral capsids. TRIM5α forms highly dynamic puncta of various sizes, and, when purified, hexagonal nets on the surface of HIV virions, but the molecular ultrastructure of the cellular bodies and the relationship of the in vitro nets to HIV restriction has remained unclear. To define the cellular ultrastructure underlying the punctate and dynamic nature of YFP-rhTRIM5α bodies, we applied cryogenic correlated light and electron microscopy combined with electron cryo-tomography to TRIM5α bodies and observed YFP-rhTRIM5α-localization to organelles found along the aggrephagy branch of the autophagy pathway. Consistent with previous work, we also found that TRIM5α forms hexagonal nets inside cells. Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy.