【前沿速递】中山大学易梅生教授和贾坤同副教授课题组揭示鱼类神经坏死病毒（NNV）致病新机制

近日，国际病原学权威期刊PLOS Pathogens在线发表了中山大学海洋科学学院易梅生教授和贾坤同副教授课题组在鱼类神经坏死病毒（NNV）受体研究方向的新进展，揭示NNV新受体HSP90ab1介导病毒入侵的分子机制，原文题为“Marine medaka heat shock protein 90ab1 is a receptor for red-spotted grouper nervous necrosis virus and promotes virus internalization through clathrin-mediated endocytosis”。

鱼类神经坏死病毒（nervous necrosis virus, NNV）是对我国养殖鱼类危害最大的病原之一，由该病毒引发的鱼类神经坏死病是我国二类动物疫病病种，对仔鱼和幼鱼的致死率可达100%，对水产养殖业造成巨大经济损失。目前尚无针对该病毒的有效防治方法。在病毒感染细胞的早期阶段，病毒首先利用其表面蛋白与宿主细胞表面的特定受体结合，随后介导病毒入侵宿主细胞并完成病毒感染。病毒与受体蛋白的结合能力决定了病毒入侵宿主细胞的效率。因此，鉴定NNV受体并揭示其入侵宿主细胞的分子机制，对于阐明NNV致病机制和发现新的抗病毒药物靶点具有重要意义。

研究团队以海水青鳉为研究对象，通过免疫共沉淀联合质谱分析的方法，发现了一批与NNV衣壳蛋白（capsid protein, CP）相互作用的宿主基因。随后在验证过程中发现海水青鳉HSP90ab1（MmHSP90ab1）蛋白和CP直接相互作用，并且MmHSP90ab1的NM域和CP的LR域是二者相互结合的核心结构域。进一步研究发现MmHSP90ab1具有细胞外膜分布特点，在NNV感染宿主的早期吸附阶段发挥重要作用，随后通过HSP90ab1抗体封闭、重组蛋白孵育和受体重建实验证明MmHSP90ab1是NNV的新受体。最后，研究者发现MmHSP90ab1和HSC70（NNV另一受体）与网格蛋白（clathrin）相互作用，促进NNV通过clathrin介导的内吞途径（clathrin -mediated endocytosis, CME）进入宿主细胞（图1）。该研究首次报道HSP90ab1是NNV的受体，揭示HSP90ab1介导NNV入侵宿主细胞的分子机制，为NNV的防治提供了潜在靶点。

图1. HSP90ab1介导NNV入侵细胞模式图

中山大学海洋科学学院张弯弯博士为该论文的第一作者，中山大学海洋科学学院易梅生教授和贾坤同副教授为该文的共同通讯作者。该研究得到了国家自然科学基金和广州市珠江科技新星等项目的资助。

附文献信息：

论文链接：

https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1008668

ABSTRACT

Nervous necrosis virus (NNV) can infect many species of fish and causes serious acute or persistent infection. However, its pathogenic mechanism is still far from clear. Specific cellular surface receptors are crucial determinants of the species tropism of a virus and its pathogenesis. Here, the heat shock protein 90ab1 of marine model fish species marine medaka (MmHSP90ab1) was identified as a novel receptor of red-spotted grouper NNV (RGNNV). MmHSP90ab1 interacted directly with RGNNV capsid protein (CP). Specifically, MmHSP90ab1 bound to the linker region (LR) of CP through its NM domain. Inhibition of MmHSP90ab1 by HSP90-specific inhibitors or MmHSP90ab1 siRNA caused significant inhibition of viral binding and entry, whereas its overexpression led to the opposite effect. The binding of RGNNV to cultured marine medaka hMMES1 cells was inhibited by blocking cell surface-localized MmHSP90ab1 with anti-HSP90β antibodies or pretreating virus with recombinant MmHSP90ab1 or MmHSP90ab1-NM protein, indicating MmHSP90ab1 was an attachment receptor for RGNNV. Furthermore, we found that MmHSP90ab1 formed a complex with CP and marine medaka heat shock cognate 70, a known NNV receptor. Exogenous expression of MmHSP90ab1 independently facilitated the internalization of RGNNV into RGNNV impenetrable cells (HEK293T), which was blocked by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Further study revealed that MmHSP90ab1 interacted with the marine medaka clathrin heavy chain. Collectively, these data suggest that MmHSP90ab1 is a functional part of the RGNNV receptor complex and involved in the internalization of RGNNV via the clathrin endocytosis pathway.

Author summary

Nervous necrosis virus (NNV), a Betanodavirus belonging to the Nodaviridae family, causes severe financial losses to aquaculture industry worldwide, nevertheless, its pathogenic mechanism remains unknown. Virus receptors are pivotal for viruses to cause infection and pathogenicity. However, knowledge regarding the cellular receptor for NNV is limited, and it has been unclear how the receptor participates in intracellular trafficking of NNV particles. In this study, we demonstrated that marine medaka heat shock protein 90ab1(MmHSP90ab1) was a functional receptor of RGNNV for the first time. Furthermore, MmHSP90ab1 expression could independently lead to RGNNV internalization through the clathrin endocytosis pathway. Our findings will contribute to developing novel strategies against NNV infection by targeting the MmHSP90ab1-clathrin endocytosis pathway.

本文来源：中山大学

本期编辑：Double