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.

中文翻译：

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伪狂犬病病毒核出口复合物-核衣壳相互作用的突变功能分析。

疱疹病毒核衣壳通过病毒核出口复合物（NEC）介导的囊泡介导的易位而离开细胞核。NEC由两个保守的病毒蛋白组成，在α疱疹病毒假狂犬病病毒（PrV）中称为pUL34和pUL31。它是有效的核出口所必需的，并且对于囊泡的形成和从内核膜（INM）的分裂都足够。基于结构的诱变鉴定出位于NEC最远膜末端的pUL31中242位（K242）处的赖氨酸，对于有效地将核衣壳掺入芽囊中至关重要。尽管在核中存在多余的核衣壳，但用丙氨酸（K242A）取代赖氨酸导致空泡囊在核周间隙中积聚。然而，尚不清楚衣壳掺入的缺陷是否是由于对衣壳与NEC之间直接的静电相互作用的干扰或结构限制引起的。为了测试这一点，我们用几种氨基酸取代了K242，从而改变了电荷，大小和侧链方向。另外，使表达pUL31-K242A的病毒重组体传代并筛选第二位点突变。鉴定出pUL31或pUL34中不同位置的补偿性突变，表明NEC具有固有的灵活性。总之，我们的数据表明，第242位的氨基酸不直接与核衣壳相互作用，但NEC外壳中的重排是有效包裹INM的有效核衣壳所必需的。重要信息疱疹病毒编码异常的囊泡形成和分裂机制，它在内核膜上运行，将病毒核衣壳从细胞核转移到核周空间。保守的疱疹病毒核出口复合物（NEC）协调了这一过程。高分辨率成像方法以及最近解决的NEC晶体结构为深入研究囊泡形成和分裂的分子细节提供了深刻的见识。然而，核衣壳掺入的分子机制仍不清楚。根据基于结构的预测，可以在NEC（pUL31-K242）的最远膜结构域中查明碱性氨基酸，这表明衣壳的掺入可能取决于直接的静电相互作用。然而，这里描述的我们的后续研究表明，正电荷无关紧要，但总体结构很重要。