Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction
Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction
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DOI：
10.1128/jvi.01910-19
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发表时间：
2020-02
影响因子：
5.4
通讯作者：
Sebastian Rönfeldt;K. Franzke;Julia E Hölper;B. Klupp;T. Mettenleiter
中科院分区：
文献类型：
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作者：
Sebastian Rönfeldt;K. Franzke;Julia E Hölper;B. Klupp;T. Mettenleiter
疱疹病毒编码了一种特殊的囊泡形成和分裂机制，它在核膜内起作用，将病毒核衣壳从细胞核转移到核周空间。保守的疱疹病毒核输出复合体（NEC）协调了这一过程。高分辨率成像方法以及最近解决的NEC晶体结构提供了对囊泡形成和分裂的分子细节的深入了解。然而，核衣壳结合的分子机制尚不清楚。根据基于结构的预测，可以在NEC的最膜远端结构域（pUL31-K242）中确定一个碱性氨基酸，这表明衣壳的结合可能依赖于直接的静电相互作用。我们的后续研究，在这里描述，然而，表明正电荷是无关的，但整体结构的问题。疱疹病毒核衣壳通过病毒核出口复合体（NEC）介导的囊泡介导的易位离开细胞核。NEC由两个保守的病毒蛋白组成，在甲型疱疹病毒伪狂犬病毒（PrV）中被命名为pUL34和pUL31。它是有效的核出口所必需的，并且足以形成囊泡和从核膜（INM）分裂。基于结构的诱变在pUL31的242 （K242）位置发现了赖氨酸，它位于NEC的大部分膜远端部分，对于核衣壳有效地整合到出芽囊泡中至关重要。用丙氨酸（K242A）代替赖氨酸导致核周空囊泡的积累，尽管细胞核中存在过量的核衣壳。然而，尚不清楚衣壳掺入缺陷是由于衣壳与NEC之间的直接静电相互作用或结构限制的干扰。为了验证这一点，我们用几个氨基酸代替了K242，从而改变了电荷、大小和侧链取向。此外，对表达pUL31-K242A的病毒重组体进行传代并筛选二位点突变。在pUL31或pUL34的不同位置发现了代偿性突变，这表明NEC具有固有的灵活性。总之，我们的数据表明，242位的氨基酸不直接与核衣壳相互作用，但NEC外壳的重排是在INM处有效包膜核衣壳所必需的。疱疹病毒编码一种特殊的囊泡形成和分裂机制，它在核膜内起作用，将病毒核衣壳从细胞核转移到核周空间。保守的疱疹病毒核输出复合体（NEC）协调了这一过程。高分辨率成像方法以及最近解决的NEC晶体结构提供了对囊泡形成和分裂的分子细节的深入了解。然而，核衣壳结合的分子机制尚不清楚。根据基于结构的预测，可以在NEC的最膜远端结构域（pUL31-K242）中确定一个碱性氨基酸，这表明衣壳的结合可能依赖于直接的静电相互作用。我们的后续研究，在这里描述，然而，表明正电荷是无关的，但整体结构的问题。
Herpesviruses encode an exceptional vesicle formation and scission machinery, which operates at the inner nuclear membrane, translocating the viral nucleocapsid from the nucleus into the perinuclear space. The conserved herpesviral nuclear egress complex (NEC) orchestrates this process. High-resolution imaging approaches as well as the recently solved crystal structures of the NEC provided deep insight into the molecular details of vesicle formation and scission. Nevertheless, the molecular mechanism of nucleocapsid incorporation remained unclear. In accordance with structure-based predictions, a basic amino acid could be pinpointed in the most membrane-distal domain of the NEC (pUL31-K242), indicating that capsid incorporation might depend on a direct electrostatic interaction. Our follow-up study, described here, however, shows that the positive charge is not relevant but that the overall structure matters. ABSTRACT Herpesvirus nucleocapsids leave the nucleus by a vesicle-mediated translocation mediated by the viral nuclear egress complex (NEC). The NEC is composed of two conserved viral proteins, designated pUL34 and pUL31 in the alphaherpesvirus pseudorabies virus (PrV). It is required for efficient nuclear egress and is sufficient for vesicle formation and scission from the inner nuclear membrane (INM). Structure-based mutagenesis identified a lysine at position 242 (K242) in pUL31, located in the most membrane distal part of the NEC, to be crucial for efficient nucleocapsid incorporation into budding vesicles. Replacing the lysine by alanine (K242A) resulted in accumulations of empty vesicles in the perinuclear space, despite the presence of excess nucleocapsids in the nucleus. However, it remained unclear whether the defect in capsid incorporation was due to interference with a direct, electrostatic interaction between the capsid and the NEC or structural restrictions. To test this, we replaced K242 with several amino acids, thereby modifying the charge, size, and side chain orientation. In addition, virus recombinants expressing pUL31-K242A were passaged and screened for second-site mutations. Compensatory mutations at different locations in pUL31 or pUL34 were identified, pointing to an inherent flexibility of the NEC. In summary, our data suggest that the amino acid at position 242 does not directly interact with the nucleocapsid but that rearrangements in the NEC coat are required for efficient nucleocapsid envelopment at the INM. IMPORTANCE Herpesviruses encode an exceptional vesicle formation and scission machinery, which operates at the inner nuclear membrane, translocating the viral nucleocapsid from the nucleus into the perinuclear space. The conserved herpesviral nuclear egress complex (NEC) orchestrates this process. High-resolution imaging approaches as well as the recently solved crystal structures of the NEC provided deep insight into the molecular details of vesicle formation and scission. Nevertheless, the molecular mechanism of nucleocapsid incorporation remained unclear. In accordance with structure-based predictions, a basic amino acid could be pinpointed in the most membrane-distal domain of the NEC (pUL31-K242), indicating that capsid incorporation might depend on a direct electrostatic interaction. Our follow-up study, described here, however, shows that the positive charge is not relevant but that the overall structure matters.