Gottwein Eva, Jäger Stefanie, Habermann Anja, Kräusslich Hans-Georg
Abteilung Virologie, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.
J Virol. 2006 Jul;80(13):6267-75. doi: 10.1128/JVI.02177-05.
The p6 domain of human immunodeficiency virus type 1 (HIV-1) Gag has long been known to be monoubiquitinated. We have previously shown that the MA, CA, and NC domains are also monoubiquitinated at low levels (E. Gottwein and H. G. Krausslich, J. Virol. 79:9134-9144, 2005). While several lines of evidence support a role for ubiquitin in virus release, the relevance of Gag ubiquitination is unclear. To directly address the function of Gag ubiquitination, we constructed Gag variants in which lysine residues in the NC, SP2, and p6 domains were mutated to arginine either in individual domains or in combination. Using these mutants, we showed that in addition to MA, CA, NC, and p6, SP2 is also mono- or di-ubiquitinated at levels comparable to those of the other domains. Replacement of all lysine residues in only one of the domains had minor effects on virus release, while cumulative mutations in NC and SP2 or in NC and p6 resulted in an accumulation of late budding structures, as observed by electron microscopy analysis. Strikingly, replacement of all lysine residues downstream of CA led to a significant reduction in virus release kinetics and a fivefold accumulation of late viral budding structures compared to wild-type levels. These results indicate that ubiquitination of lysine residues in Gag in the vicinity of the viral late domain is important for HIV-1 budding, while no specific lysine residue may be needed and individual domains can functionally substitute. This is consistent with Gag ubiquitination being functionally involved in a transient protein interaction network at the virus budding site.
长期以来，人们一直知道1型人类免疫缺陷病毒（HIV-1）Gag的p6结构域会发生单泛素化。我们之前已经表明，MA、CA和NC结构域在低水平时也会发生单泛素化（E. Gottwein和H. G. Krausslich，《病毒学杂志》79:9134 - 9144，2005年）。虽然有几条证据支持泛素在病毒释放中起作用，但Gag泛素化的相关性尚不清楚。为了直接探讨Gag泛素化的功能，我们构建了Gag变体，其中NC、SP2和p6结构域中的赖氨酸残基在单个结构域或组合中被突变为精氨酸。使用这些突变体，我们表明，除了MA、CA、NC和p6之外，SP2也会发生单泛素化或双泛素化，其水平与其他结构域相当。仅在一个结构域中替换所有赖氨酸残基对病毒释放的影响较小，而NC和SP2或NC和p6中的累积突变导致晚期出芽结构的积累，这通过电子显微镜分析观察到。令人惊讶的是，与野生型水平相比，CA下游所有赖氨酸残基的替换导致病毒释放动力学显著降低，晚期病毒出芽结构积累了五倍。这些结果表明，病毒晚期结构域附近Gag中赖氨酸残基的泛素化对于HIV-1出芽很重要，而可能不需要特定的赖氨酸残基，并且各个结构域可以在功能上相互替代。这与Gag泛素化在功能上参与病毒出芽位点的瞬时蛋白质相互作用网络是一致的。
