Rab蛋白调控猪瘟病毒入胞后的转运以及参与核衣壳释放的分子机制研究

Rab proteins are a large family of small GTPases that serve as multifaceted organizers of almost all cellular membrane trafficking processes. Studies have shown that viruses can hijack several Rab proteins (including 3,5,7,9 and 11) to facilitate their intracellular trafficking and genome replication. Previously we have reported that clathrin-dependent entry of classical swine fever virus (CSFV) into PK-15 cells requires Rab5 and Rab7. However, the exact role of Rab proteins, particularly the Rab5 and Rab7, in the CSFV entry process and the subsequent viral neucleocaspid uncoating are unknown. Therefore, the goal of this project is to investigate the defined roles of Rab protein family in regulating CSFV infection after entering its natural host cells and to understand the molecular mechanisms of CSFV endocytosis, membrane fusion, and nucleocapsid release into cytosol. Understanding the early virus entry and uncoating pathway will help us to design target-specific antiviral drugs. We have five specific aims for this project. 1). Identify Rab proteins that regulate early endosome, later endosome and lysosome pathway during CSFV infection. 2). Study whether acid environment will promote fusion between endosomal membrane and CSFV viral envelope membranes. 3). Study the role of intracellular effect proteins associated with Rab proteins. 4). Investigate the role of cytoskeleton (microfilaments and microtubules) in CSFV intracellular trafficking. 5). Identify the key domains of CSFV envelope glycoprotein that may contribute to endosomal membrane fusion. This project will greatly enhance our understanding of the role of Rab proteins in CSFV infection, which has not been well characterized yet.

Rab蛋白是细胞囊膜运输途径关键调节因子，调控从膜转运囊泡形成到靶向释放的全过程，已有研究表明Rab家族主要成员（3、5、7、9和11等）参与了病毒侵染宿主细胞的各个过程。近期我们报道了网格状蛋白介导猪瘟病毒（CSFV）进入猪肾细胞（PK-15）后，需要Rab5和Rab7调控囊泡运输病毒颗粒，但是其下游效应分子以及调控CSFV入胞后释放核衣壳的分子机制尚不清楚。聚焦这些问题，本研究拟开展：1）鉴定参与调控内体和溶酶体功能，协助CSFV感染的Rab蛋白；2)研究酸性环境对内体膜与病毒囊膜的膜融合的影响；3）鉴定Rab蛋白招募的效应因子及其功能；4）研究细胞骨架（微丝/微管）协助CSFV入胞后的迁移；5）鉴定病毒囊膜糖蛋白参与膜融合的关键位点。该研究将填补瘟病毒入胞及其病毒与宿主细胞互作的相关研究空白，有利于开发抗病毒药物。

Rab蛋白是细胞囊膜运输途径的关键调节因子。虽然已有研究表明Rab家族多个成员参与调控病毒侵染细胞的各个过程，然而他们在猪瘟病毒（CSFV）侵染过程中发挥的功能及其调控机制未有报道。因此，本研究目的是鉴定参与调控Rab蛋白及其招募的效应因子在CSFV侵染靶细胞分子过程中发挥的作用。本项目已经开展了：（1）Rab蛋白调控CSFV侵染猪肺泡巨噬细胞的机制研究；（2）细胞骨架调控CSFV侵染细胞的机制研究；（3）分子马达在CSFV侵染中发挥的作用及其机制；（4）Rab蛋白调控CSFV脱衣壳的机制研究；（5）研究Rab蛋白调控CSFV感染细胞后的脂代谢重编程；（6）内吞体分选转运复合体（ESCRT）调控CSFV入侵细胞的机制研究。针对上述6个主要研究内容，实施项目后，获得如下成果：（1）揭示了Rab5/7/11在CSFV以小窝蛋白介导感染猪肺泡巨噬细胞中逐级运输病毒至溶酶体的分子机制；（2）研究发现了细胞骨架成分微丝、微管和波形蛋白交替协调CSFV侵染细胞的多步内体转运过程。（3）阐明了分子马达Dynein和Kinesin-1调控CSFV沿乙酰化微管的胞内运输至溶酶体的分子机制。（4）揭示了含缬酪肽蛋白（VCP）在Rab蛋白帮助下在内质网诱导CSFV脱衣壳的分子机制。（5）阐明了脂肪酸合成酶与NS4B的互作促进病毒复制的分子机制。（6）揭示了Rab14调节神经酰胺从内质网向高尔基体的运输并促进病毒组装的分子机制。（7）鉴定了ESCRT-I主要亚基Tsg101在CSFV入胞和复制中的双重作用。总之，我们的研究详细地阐述了CSFV在入胞到病毒脱衣壳过程中，Rab蛋白家族的多个成员参与调控的过程，上述靶点均可以作为抗病毒靶标，有利于抗病毒药物的创制。