Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phutthamonthon Sai 4 Road, Salaya, Nakhon Pathom, 73170, Thailand.
Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.
Sci Rep. 2024 May 6;14(1):10407. doi: 10.1038/s41598-024-61195-z.
Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.
葡萄糖调节蛋白 78（GRP78）是一种伴侣蛋白，是未折叠蛋白反应的中心介质，是一种关键的细胞应激反应途径。已经表明，GRP78 对于多种黄病毒的感染和复制是至关重要的，并且与非结构（NS）和结构黄病毒蛋白相互作用。然而，GRP78 与病毒蛋白之间的特定相互作用的性质在很大程度上仍然未知。本研究旨在表征结合结构域和关键氨基酸残基，介导 GRP78 与 ZIKV E 和 NS1 蛋白的相互作用。用重组 EGFP 融合的 GRP78 和各个亚域（核苷酸结合域（NBD）和底物结合域（SBD））作为诱饵蛋白，并与全长或截短的 ZIKV E 和 NS1 蛋白在 HEK293T/17 细胞中共表达。通过共免疫沉淀测定蛋白质-蛋白质相互作用。结果表明，GRP78 的 NBD 和 SBD 对于有效的相互作用都是至关重要的。SBD 中的单个氨基酸取代表明，R492E 和 T518A 突变体显著降低了 GRP78 与 ZIKV E 和 NS1 蛋白的结合亲和力。值得注意的是，GRP78 与 ZIKV E 的相互作用在 ZIKV E 结构域 III 上的各种单个氨基酸取代和所有截短的 ZIKV E 和 NS1 蛋白中都保持稳定。总之，结果表明，GRP78 与病毒蛋白之间的主要结合主要是一种经典的典型伴侣蛋白-客户相互作用。阻断 GRP78 伴侣功能可有效抑制神经祖细胞中的 ZIKV 感染和复制。我们的研究结果表明，GRP78 是抗 ZIKV 治疗的潜在宿主靶标。
