K63-Linked Ubiquitin Is Required for Restriction of HIV-1 Reverse Transcription and Capsid Destabilization by Rhesus TRIM5α

TRIM5 alpha is an antiviral restriction factor that inhibits retroviral infection in a species-specific fashion. TRIM5 alpha binds to and forms assemblies around the retroviral capsid. Following binding, poorly understood, ubiquitin-dependent events lead to the disassembly of the viral core, prior to the accumulation of viral reverse transcription products in the target cell. It is also known that assemblies of TRIM5 alpha and other TRIM family proteins can be targets of autophagic degradation. The goal of this study was to define the role of specific ubiquitin linkages in the retroviral restriction and autophagic degradation of TRIM5 alpha and delineate any connection between these two processes. To this end, we generated fusion proteins in which the catalytic domains of different deubiquitinase (DUB) enzymes, with different specificities for polyubiquitinated linkages, were fused to the N-terminal RING domain of Rhesus macaque TRIM5 alpha. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5 alpha with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5 alpha is required to induce capsid disassembly and to inhibit reverse transcription of HIV, while the ability to inhibit HIV-1 infection was not dependent on K63-linked ubiquitination. We also observed that K63-linked ubiquitination is required for the association of TRIM5 alpha with autophagosomal membranes and the autophagic adapter protein p62. IMPORTANCE Although the mechanisms by which TRIM5 alpha can induce the abortive disassembly of retroviral capsids have remained obscure, numerous studies have suggested a role for ubiquitination and cellular degradative pathways. These studies have typically relied on global perturbation of cellular degradative pathways. Here, through the use of linkage-specific deubiquitinating enzymes tethered to TRIM5 alpha, we delineate the ubiquitin linkages which drive specific steps in restriction and degradation by TRIM5 alpha, providing evidence for a noncanonical role for K63-linked ubiquitin in the process of retroviral restriction by TRIM5 alpha and potentially providing insight into the mechanism of action of other TRIM family proteins.