Molecular Basis for Nuclear Egress of Herpesviruses

疱疹病毒核衣壳通过在内核膜获得初级包膜，随后与外核膜融合，从其在核中的组装位点转移到胞质溶胶。这种通过核膜的囊泡运输货物在细胞生物学中是新颖的。它需要保守的疱疹病毒pUL 31和pUL 34蛋白的同源物的存在，这些蛋白形成核出口复合物（NEC）。NEC募集病毒和细胞激酶以局部溶解核纤层，从而允许核衣壳进入内核膜并与NEC相互作用，然后通过核衣壳周围的NEC的寡聚化来驱动初级降解。然而，目前还不清楚货物是如何被招募到囊泡，它们的裂变是如何发生的，特别是，是什么驱动它们与外核膜过程的融合，其中可能不仅包括病毒，还包括细胞蛋白。虽然在第一阶段，我们专注于阐明α疱疹病毒NEC的功能和结构，但我们现在希望继续分析其寡聚化，从而导致囊泡形成，探索通过冷冻-EM重建预测的相互作用表面;其在货物中的活性，通过预测的NEC-衣壳相互作用位点的突变的（核衣壳）识别;通过CRISPR/Cas9诱变鉴定参与融合的潜在细胞蛋白，包括作为对照病毒突变体，其通过核膜破裂离开细胞核，而不依赖于已经在第一阶段通过回复分析分离的NEC;并在异疱疹病毒科中筛选NEC的结构和功能同源物，其不显示显著的序列保守性，但核出口途径保守。这些研究不仅揭示了疱疹病毒核出口的分子基础，而且还确定了参与这种独特的囊泡核质转运的细胞蛋白。

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.