Department of Microbiology & Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794-5222, USA.
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Viruses. 2020 Apr 15;12(4):447. doi: 10.3390/v12040447.
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-ΔZnF2-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.
ESCRT-I 因子 Tsg101 对于分拣内吞货物是必不可少的，并且被病毒病原体利用来促进从细胞中逸出。HIV-1 Gag 中的核衣壳 (NC) 结构域和 p6 结构域都有助于该蛋白的募集。然而，当 p6 中的 P(S/T)AP 基序完整时，NC 的作用尚不清楚，因为该基序可以直接募集 Tsg101。NC 中的锌指结合 RNA 和膜，对于出芽至关重要。Tsg101 可以替代远端 ZnF（ZnF2）并挽救因该元件缺失而导致缺陷的突变体的出芽。在这里，我们报告 Tsg101 的泛素 (Ub) E2 变体 (UEV) 结构域在体外与 tRNA 结合。我们证实，当作为嵌合 Gag-Tsg101 蛋白（Gag-ΔZnF2-Tsg101）提供在病毒组装部位时，Tsg101 可以替代 ZnF2 并挽救出芽。在这种情况下，UEV 并不需要；然而，当 Gag 和 Tsg101 共表达时，UEV 中的 RNA 结合决定因素的突变阻止了 Tsg101 从细胞内部募集。相同的 Tsg101 突变增加了对 Gag-Ub 的识别，表明 tRNA 和 Ub 竞争结合位点。这项研究确定了一种新的 Tsg101 结合伙伴，它可能有助于其识别 Ub 修饰货物的功能。
