Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.
Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.
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DOI：
10.1371/journal.ppat.1001153
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发表时间：
2010-10-21
期刊：
影响因子：
6.7
通讯作者：
Bieniasz PD
中科院分区：
文献类型：
--
作者：
Zhadina M;Bieniasz PD
将新生包膜病毒体与宿主细胞膜分离的膜断裂事件通常需要ESCRT途径，其可以通过病毒蛋白中称为晚期（L-）结构域的肽基序的作用参与。病毒PTAP和YPDL样L结构域直接结合ESCRT途径的ESCRT-I和阿利克斯组分，而PPxY基序结合Nedd 4样、含HECT结构域的泛素连接酶（例如WWP 1）。目前还不清楚泛素连接酶募集最终如何导致颗粒释放。在这里，我们使用源自原型泡沫病毒（PFV）的无赖氨酸病毒Gag蛋白（其中可以控制泛蛋白与Gag的附着），表明几种不同的HECT结构域可以取代嵌合泛蛋白连接酶中的WWP 1 HECT结构域并驱动萌芽。此外，将分离的HECT结构域人工募集至Gag足以刺激出芽。相反，如果WWP 1的其他结构域直接融合到ESCRT-1蛋白，则HECT结构域变为双链。在每种情况下，出芽是由HECT结构域驱动，其催化活性是必不可少的，但Gag泛素化是不必要的，这表明泛素连接到反式作用蛋白驱动出芽。然而，我们也证明了泛素部分与PFV Gag的C-末端的直接融合也可以促进出芽，这表明Gag的泛素化可以取代反式作用蛋白的泛素化。Tsg 101和阿利克斯的耗尽抑制依赖于与Gag融合或通过PPxY基序的作用与反式作用蛋白连接的泛素的出芽。这些研究强调了ESCRT途径可以参与的方式的灵活性，并提出了一种模型，其中泛素所连接的蛋白质的身份对于ESCRT途径的泛素结合组分的随后招募和病毒出芽进行并不重要。包膜病毒颗粒从感染细胞中释放需要分离病毒和细胞膜。许多包膜病毒通过寄生一组称为ESCRT途径的细胞蛋白来实现这一点，该途径通常将细胞膜彼此分离。在某些情况下，病毒结构蛋白编码直接结合并从而募集ESCRT机制的肽基序。或者，病毒可以募集与其他蛋白质结合的酶，称为泛素连接酶，并催化泛素与它们的结合。迄今为止，还不清楚泛素连接酶的募集是如何导致ESCRT机制的参与的。我们表明，简单的招聘的一个片段的泛素连接酶，负责添加的泛素到其他蛋白质是足以驱动病毒颗粒释放，即使它是不可能的连接泛素的病毒蛋白。有趣的是，我们还发现，泛素与相同病毒蛋白的简单连接也可以驱动颗粒释放。这些结果表明，在ESCRT机制可以被招募的方式以及泛素如何被选择以实现这一点方面存在灵活性。
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed. The release of an enveloped virus particle from an infected cell requires the separation of the viral and cell membranes. Many enveloped viruses accomplish this by parasitizing a set of cellular proteins, termed the ESCRT pathway, that normally separates cellular membranes from each other. In some cases, viral structural proteins encode peptides motifs that bind directly to, and thereby recruit, the ESCRT machinery. Alternatively, viruses can recruit enzymes, termed ubiquitin ligases, that bind to other proteins, and catalyze the addition of ubiquitin to them. It has, heretofore, been somewhat unclear precisely how the recruitment of ubiquitin ligases leads to the engagement of the ESCRT machinery. We show that the simple recruitment of a fragment of a ubiquitin ligase that is responsible for the addition of ubiquitin to other proteins is sufficient to drive virus particle release, even when it is not possible to attach ubiquitin to viral proteins. Paradoxically, we also found that simple attachment of ubiquitin to the same viral protein can also drive particle release. These results show that there is flexibility in the ways in which the ESCRT machinery can be recruited and how ubiquitin can be co-opted to enable this.
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DOI：
10.1126/science.1161070
发表时间：
2008-09-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Lata S;Schoehn G;Jain A;Pires R;Piehler J;Gottlinger HG;Weissenhorn W
通讯作者：
Weissenhorn W
影响因子：
64.5
作者：
Katzmann, DJ;Babst, M;Emr, SD
通讯作者：
Emr, SD
影响因子：
3.3
作者：
Bishop, N;Woodmane, P
通讯作者：
Woodmane, P
DOI：
10.1083/jcb.200408155
发表时间：
2005-01-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Martin-Serrano J;Eastman SW;Chung W;Bieniasz PD
通讯作者：
Bieniasz PD
影响因子：
16.8
作者：
Alam, Steven L.;Langelier, Charles;Sundquist, Wesley I.
通讯作者：
Sundquist, Wesley I.