RNA Binding Suppresses Tsg101 Recognition of Ub-Modified Gag and Facilitates Recruitment to the Plasma Membrane

Abstract

The ESCRT-I factor Tsg101 is essential for sorting endocytic cargo and is exploited by viral pathogens to facilitate egress from cells. Both the nucleocapsid (NC) domain and p6 domain in HIV-1 Gag contribute to recruitment of the protein. However, the role of NC is unclear when the P(S/T)AP motif in p6 is intact, as the motif recruits Tsg101 directly. The zinc fingers in NC bind RNA and membrane and are critical for budding. Tsg101 can substitute for the distal ZnF (ZnF2) and rescue budding of a mutant made defective by deletion of this element. Here, we report that the ubiquitin (Ub) E2 variant (UEV) domain in Tsg101 binds tRNA in vitro. We confirmed that Tsg101 can substitute for ZnF2 when provided at the viral assembly site as a chimeric Gag-Tsg101 protein (Gag-∆ZnF2-Tsg101) and rescue budding. The UEV was not required in this context; however, mutation of the RNA binding determinants in UEV prevented Tsg101 recruitment from the cell interior when Gag and Tsg101 were co-expressed. The same Tsg101 mutations increased recognition of Gag-Ub, suggesting that tRNA and Ub compete for binding sites. This study identifies a novel Tsg101 binding partner that may contribute to its function in recognition of Ub-modified cargo.

Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role

“…The p6 domain is known to be involved in the recruitment of host factors, such as Tsg101 (Tumor susceptibility gene 101) and ALIX (ALG-2 interacting protein X), and ubiquitination of those factors strongly promote viral budding [ 135 ]. In this frame, fusion experiments in which the p6 domain was coupled with Ub showed that the affinity of Tsg101 for p6 in this case results in being strengthened [ 136 ], and the ubiquitination of Pr55 Gag can increase Tsg101 recruitment [ 137 ]. Besides, Tsg101 displays an N-terminal Ub E2 variant (UEV) domain that shows homology with E2 Ub ligases, and that can specifically bind Ub proteins, as well the PTAP late domain in Pr55 Gag [ 136 ].…”

“…Besides, Tsg101 displays an N-terminal Ub E2 variant (UEV) domain that shows homology with E2 Ub ligases, and that can specifically bind Ub proteins, as well the PTAP late domain in Pr55 Gag [ 136 ]. During assembly, the interaction of Pr55 Gag with the PM promotes the intermolecular interaction between Tsg101 and the PTAP domain in Pr55 Gag [ 137 ]. In this conformation, the di-ubiquitinylated K63 of Tsg101 was found to interact with p6, with the consequence of impairing the potential polyubiquitination of the precursor at PM [ 137 ].…”

“…During assembly, the interaction of Pr55 Gag with the PM promotes the intermolecular interaction between Tsg101 and the PTAP domain in Pr55 Gag [ 137 ]. In this conformation, the di-ubiquitinylated K63 of Tsg101 was found to interact with p6, with the consequence of impairing the potential polyubiquitination of the precursor at PM [ 137 ].…”

How HIV-1 Gag Manipulates Its Host Cell Proteins: A Focus on Interactors of the Nucleocapsid Domain

“…This polymerization activates VPS-4, an ATP free hydrolase, which in turn disassembles the ESCRT-III domain by hydrolyzing its proteins leading to the pinching off of the virion from the PM [172]. A recent study has reported that the proper recruitment of Tsg101 by PTAP is further controlled by RNA (probably bound to NC domain) and that the latter prevents Gag polyubiquitination and thus its degradation [183].…”

Novel Tsg101 Binding Partners Regulate Viral L Domain Trafficking

“…The UEV domain mediates these Tsg101 functions through the P(T/S)AP-and Ub-binding functions. The region downstream of the UEV domain in Tsg101 interacts with other proteins, including the binding partners with which it forms ESCRT-I, a complex that functions with ESCRT-0, -II, -III, and the ATPase Vps4 in endocytic trafficking (reviewed in [4,6,14,15]). As the member on which the partners nucleate, Tsg101 thus controls ESCRT-I formation and plays an essential scaffolding and mechanical role in addition to functioning as a conduit to ESCRT-III [8].…”