伪狂犬病毒核输出复合体摄取核衣壳的作用机制研究

Pseudorabies, a highly contagious infectious disease of swine, is caused by pseudorabies virus (PRV). Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. PRV is often used as a model system to study alphaherpesvirus infections. Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane, as well as from synthetic lipid bilayers. However, how cargo is incorporated remained unclear. In this proposal, we plan to study the mechanism of this special uptake and try to solve the three-dimensional structure of the complex of NEC and proteins on capsid. In the meantime the structural study will uncover the interaction mode between NEC and capsid. The proposed study will not only improve our understanding of the alphaherpesvirus replication process, but also provide a basis for further developing novel antivirus drugs.

伪狂犬病是一种由猪伪狂犬病毒引起的高度接触性传染病。自2011年以来，高致病性伪狂犬病病毒抗原变异毒株在中国肆虐，造成了巨大的经济损失。新组装的疱疹病毒核衣壳通过囊泡介导机制出核膜转移到细胞质中。这个过程由α-疱疹病毒保守性核输出复合物NEC控制，它由pUL31和pUL34的蛋白同系物组成。NEC的功能可以形成有效的核出口，还能够介导内核膜摄取核衣壳形成脂质双层囊泡。然而核衣壳如何被摄取仍不清楚。在本次申请计划中，我们计划研究这种特殊的摄取机制，尝试揭示NEC与核衣壳表面特定蛋白复合物的三维结构，揭示出NEC与核衣壳之间的相互作用模式。本项研究不仅能够完善对α-疱疹病毒复制过程的理解，也为进一步抗病毒药物靶点的选择提供理论依据。

伪狂犬病是一种由猪伪狂犬病毒引起的高度接触性传染病。自2011年以来高致病性伪狂犬病病毒抗原变异毒株在中国肆虐，造成了巨大的经济损失。新生子代疱疹病毒核衣壳通过囊泡介导出核膜转移到细胞质中。这个过程由α-疱疹病毒亚家族保守的核输出复合物NEC控制，它由pUL31和pUL34的蛋白同系物组成。NEC的功能可以形成有效的核出口，介导内核膜摄取核衣壳形成脂质双层囊泡。然而在此过程中，核衣壳如何被摄取仍不清楚。本项目中研究这种特殊的摄取机制，尝试通过体外同源重组方法分别表达纯化出NEC与核衣壳表面特定蛋白复合物，并研究复合物组装及其三维结构，揭示出NEC与核衣壳之间的相互作用模式。本项目首次建立了全长NEC复合体以及衣壳顶端特定成分CVSC中pUL17-pUL25复合体的体外组装纯化方法，并初步尝试组装NEC-pUL17-pUL25复合物用于结构解析。目前已经解析全长pUL17-pUL25的低分辨率结构。但仍需改进纯化方法与条件，提高pUL17-pUL25复合体的分辨率以及解析出NEC-pUL17-pUL25复合物结构。这些成果将促进对α-疱疹病毒复制过程的理解。