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Molecular Basis for Nuclear Egress of Herpesviruses

疱疹病毒核排出的分子基础

基本信息

批准号：
226088690
负责人：
Professor Dr. Thomas C. Mettenleiter
金额：
--
依托单位：
Institut für molekulare Virologie und Zellbiologie (IMVZ)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/226088690?language=en
关键词：
Molecular Basis Nuclear Egress Herpesviruses

项目摘要

Herpesvirus nucleocapsids are translocated from their assembly site in the nucleus to the cytosol by acquisition of a primary envelope at the inner nuclear membrane which subsequently fuses with the outer nuclear membrane. This vesicular transport of cargo through the nuclear envelope is novel in cell biology. It requires the presence of homologs of the conserved herpesviral pUL31 and pUL34 proteins which form the nuclear egress complex (NEC). The NEC recruits viral and cellular kinases to locally dissolve the nuclear lamina allowing access of nucleocapsids to the inner nuclear membrane and interaction with the NEC, which then drives primary envelopment by oligomerization of the NEC around the nucleocapsid. However, it is still unclear how cargo is recruited to the vesicle, how their fission occurs and, in particular, what drives their fusion with the outer nuclear membrane processes which may include not only viral but also cellular proteins. While in the first period we focussed on the elucidation of function and structure of the alphaherpesvirus NEC, we now want to continue by analysis of its oligomerization leading to vesicle formation probing for interacting surfaces predicted by the cryo-EM reconstruction; its activity in cargo (nucleocapsid) recognition by mutation of predicted NEC-capsid interacting sites; identify potential cellular proteins involved in fusion by CRISPR/Cas9 mutagenesis including as control virus mutants which leave the nucleus by nuclear envelope breakdown independent of the NEC which had been isolated in the first period by reversion analysis; and screen for structural and functional homologs of the NEC in the Alloherpesviridae which do not show significant sequence conservation but conservation of the nuclear egress pathway. These studies should not only shed more light on the molecular basis of herpesvirus nuclear egress but also identify cellular proteins involved in this unique vesicular nucleo-cytoplasmic transport.

疱疹病毒核衣壳通过在核内膜获得一个初级包膜，然后与外核膜融合，从其在核内的组装位置转移到胞浆。这种通过核膜的囊泡运输在细胞生物学中是新的。它需要存在保守的疱疹病毒pUL31和pUL34蛋白的同源物，它们形成核出口复合体(NEC)。NEC招募病毒和细胞激酶来局部溶解核膜，允许核衣壳进入内核膜并与NEC相互作用，然后NEC通过NEC围绕核衣壳的寡聚化来驱动初级包膜。然而，目前仍不清楚货物是如何被招募到囊泡中的，它们是如何发生裂变的，尤其是什么驱动它们与外部核膜过程融合，这些过程可能不仅包括病毒，还可能包括细胞蛋白质。虽然在第一阶段我们专注于阐明甲型疱疹病毒NEC的功能和结构，但我们现在想继续通过分析其寡聚导致囊泡形成的作用来探索冷冻-EM重建预测的相互作用表面；它在通过预测的NEC-衣壳相互作用位点的突变而识别货物(核衣壳)中的活性；通过CRISPR/Cas9突变鉴定参与融合的潜在细胞蛋白，包括作为对照的病毒突变体，这些突变体通过核膜破裂离开细胞核，而不依赖于第一阶段分离的NEC；以及筛选异种疱疹病毒科中NEC的结构和功能同源基因，这些结构和功能同源基因没有显示显著的序列保守，但核出口途径保守。这些研究不仅应该阐明疱疹病毒核外流的分子基础，而且还应该识别参与这种独特的囊泡核质运输的细胞蛋白。