Functional Replacement of a Retroviral Late Domain by Ubiquitin Fusion
Functional Replacement of a Retroviral Late Domain by Ubiquitin Fusion
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DOI：
10.1111/j.1600-0854.2008.00817.x
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发表时间：
2008-11-01
期刊：
影响因子：
4.5
通讯作者：
Freed, Eric O.
中科院分区：
文献类型：
--
作者：
Joshi, Anjali;Munshi, Utpal;Freed, Eric O.
逆转录病毒Gag多蛋白前体是病毒样颗粒(VLP)从感染细胞组装和释放的必要条件和充分条件。众所周知，被称为晚期结构域的GAG编码的小基序通过与细胞内体分选和泛素化机制的组件相互作用来促进颗粒释放。许多不同逆转录病毒的Gag蛋白是泛素化的；然而，Gag泛素化在颗粒出口中的作用尚不清楚。在这项研究中，我们使用一组马传染性贫血病毒(EIAV)Gag衍生物来研究这个问题，这些衍生物含有野生型EIAV晚期结构域、异源逆转录病毒晚期结构域或没有晚期结构域。将泛素融合到这些Gag多聚蛋白的C末端，并检测其对VLP萌发的影响。值得注意的是，泛素与缺乏晚期结构域的EIAV Gag(EIAV/Delta YPDL-Ub)的融合在很大程度上挽救了VLP的释放。我们还确定了泛素融合对颗粒释放对萌发抑制物和关键内体分选因子耗尽的敏感性的影响。泛素融合使EIAV/Delta YPDL-Ub对细胞内体分选因子Tsg101和Alix的缺失以及Tsg101和Alix的显性-阴性片段的过度表达敏感。这些发现表明，泛素可以在功能上弥补逆转录病毒晚期结构域的缺失，并提供对泛素在颗粒外泄过程中参与的宿主细胞机制的洞察。
Retroviral Gag polyprotein precursors are both necessary and sufficient for the assembly and release of virus-like particles (VLPs) from infected cells. It is well established that small Gag-encoded motifs, known as late domains, promote particle release by interacting with components of the cellular endosomal sorting and ubiquitination machinery. The Gag proteins of a number of different retroviruses are ubiquitinated; however, the role of Gag ubiquitination in particle egress remains undefined. In this study, we investigated this question by using a panel of equine infectious anemia virus (EIAV) Gag derivatives bearing the wild-type EIAV late domain, heterologous retroviral late domains or no late domain. Ubiquitin was fused in cis to the C-termini of these Gag polyproteins, and the effects on VLP budding were measured. Remarkably, fusion of ubiquitin to EIAV Gag lacking a late domain (EIAV/Delta YPDL-Ub) largely rescued VLP release. We also determined the effects of ubiquitin fusion on the sensitivity of particle release to budding inhibitors and to depletion of key endosomal sorting factors. Ubiquitin fusion rendered EIAV/Delta YPDL-Ub sensitive to depletion of cellular endosomal sorting factors Tsg101 and Alix and to overexpression of dominant-negative fragments of Tsg101 and Alix. These findings demonstrate that ubiquitin can functionally compensate for the absence of a retroviral late domain and provide insights into the host-cell machinery engaged by ubiquitin during particle egress.