Xu Xingang, Liu Yi, Gao Jie, Shi Xiaojie, Yan Yuchao, Yang Naling, Wang Quanqiong, Zhang Qi
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
Vet Microbiol. 2023 Apr;279:109666. doi: 10.1016/j.vetmic.2023.109666. Epub 2023 Jan 24.
Porcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV) has caused huge losses in the swine industry worldwide. Glucosyltransferase Rab-like GTPase activator and myotubularin domain containing 4 (GRAMD4) is a proapoptotic protein, which replaced p53 inducing mitochondrial apoptosis. However, the relationship between GRAMD4 and PEDV has not been reported. Here, we aimed to investigate the potential role of GRAMD4 during PEDV infection. In this study, we used co-immunoprecipitation (co-IP) and mass spectrometry to identify GRAMD4 interaction with PEDV non-structural protein 6 (NSP6). Immunoprecipitation and laser confocal microscopy were utilized to demonstrate that GRAMD4 interacts with NSP6. NSP6 reduces GRAMD4 production through PERK and IRE1 pathway-mediated apoptosis. We demonstrated that overexpression of GRAMD4 effectively impaired the replication of PEDV, whereas knockdown of GRAMD4 facilitated the replication of PEDV. Overexpression of GRAMD4 increased GRP78, phosphorylated PERK (p-PERK), phosphorylated IRE1(p-IRE1) levels, promoted CHOP, phosphorylated JNK (p-JNK), Bax expression, caspase 9 and caspase 3 cleavage, and inhibited Bcl-2 production. Knockdown of GRAMD4 has the opposite effect. Finally, deletion of the GRAM domain of GRAMD4 cannot cause endoplasmic reticulum stress (ER stress)-mediated apoptosis and inhibit virus replication. In conclusion, these studies revealed the mechanism by which GRAMD4 was associated with ER stress and apoptosis regulating PEDV replication. NSP6 acted as a potential down-regulator of GRAMD4 and promoted the degradation of GRAMD4. GRAMD4 played a role in facilitating apoptosis and restricting virus replication, and the GRAM domain was required. These findings provided a reference for host-PEDV interactions and offered the possibility for PEDV decontamination and prevention.
由猪流行性腹泻病毒（PEDV）引起的猪流行性腹泻（PED）给全球养猪业造成了巨大损失。含葡萄糖基转移酶Rab样GTPase激活剂和肌管素结构域4（GRAMD4）是一种促凋亡蛋白，它替代p53诱导线粒体凋亡。然而，GRAMD4与PEDV之间的关系尚未见报道。在此，我们旨在研究GRAMD4在PEDV感染过程中的潜在作用。在本研究中，我们利用免疫共沉淀（co-IP）和质谱鉴定GRAMD4与PEDV非结构蛋白6（NSP6）的相互作用。采用免疫沉淀和激光共聚焦显微镜证实GRAMD4与NSP6相互作用。NSP6通过PERK和IRE1途径介导的凋亡降低GRAMD4的产生。我们证明，GRAMD4的过表达有效损害PEDV的复制，而GRAMD4的敲低则促进PEDV的复制。GRAMD4的过表达增加了GRP78、磷酸化PERK（p-PERK）、磷酸化IRE1（p-IRE1）水平，促进了CHOP、磷酸化JNK（p-JNK）、Bax表达、caspase 9和caspase 3的裂解，并抑制了Bcl-2的产生。GRAMD4的敲低则产生相反的效果。最后，GRAMD4的GRAM结构域缺失不会引起内质网应激（ER应激）介导的凋亡，也不会抑制病毒复制。总之，这些研究揭示了GRAMD4与ER应激及凋亡相关调节PEDV复制的机制。NSP6作为GRAMD4的潜在下调因子，促进GRAMD4的降解。GRAMD4在促进凋亡和限制病毒复制中发挥作用，且需要GRAM结构域。这些发现为宿主与PEDV的相互作用提供了参考，并为PEDV的净化和预防提供了可能性。
