VP40 octamers are essential for Ebola virus replication
VP40 octamers are essential for Ebola virus replication
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DOI：
10.1128/jvi.79.3.1898-1905.2005
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发表时间：
2005-02-01
影响因子：
5.4
通讯作者：
Weissenhorn, W
中科院分区：
文献类型：
--
作者：
Hoenen, T;Volchkov, V;Weissenhorn, W
埃博拉病毒的基质蛋白VP 40是病毒组装和出芽所必需的。单体VP 40可以在体外寡聚成RNA结合的八聚体，并且八聚体VP 40的晶体结构已经揭示残基Phe 125和Arg 134是短单链RNA配位的最重要残基。在这里，我们表明，全长野生型VP 40八聚体结合RNA后HEK 293细胞表达。虽然Phe 125-to-Ala突变导致RNA结合减少，但Arg 134-to-Ala突变完全消除了RNA结合，从而消除了八聚体形成。然而，八聚体形成的缺乏不影响病毒样颗粒（VLP）形成，因为在HEK 293细胞中由野生型VP 40和突变型VP 40的表达产生的VLP显示出相似的形态和丰度，并且在大小上没有显著差异。这些结果强烈地表明八聚体VP 40对于VLP形成是不稳定的。突变体VP 40的细胞定位与野生型VP 40不同。虽然野生型VP 40主要存在于质膜上的小斑块中，但八聚体阴性突变体存在于细胞周边和核周区域的较大聚集体中。接下来，我们将Arg 134-to-Ala和/或Phe 125-to-Ala突变引入埃博拉病毒基因组。重组野生型病毒和表达VP 40 Phe 125-to-Ala突变的病毒均被拯救。相反，不能回收表达VP 40 Arg 134-to-Ala突变的重组病毒。这些结果表明，VP 40的RNA结合以及因此形成的八聚体对于埃博拉病毒的生命周期是必不可少的。
Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle.