CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
J Neuroinflammation. 2018 Sep 21;15(1):275. doi: 10.1186/s12974-018-1311-5.
Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo.
Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo.
ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased.
Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.
许多病毒依赖内质网（ER）广泛的膜网络来进行翻译、复制和包装。内质网的某些膜修饰可能会引发内质网应激，以及病毒感染导致 ER 中病毒蛋白的积累。然后，未折叠蛋白反应（UPR）被激活以减轻应激。寨卡病毒（ZIKV）是一种蚊媒黄病毒，其感染会导致新生儿小头畸形和成人严重神经并发症。在这里，我们研究了 ZIKV 在体外和体内感染神经细胞中的内质网应激和 UPR 调节模型。
通过腹腔注射感染 I 型和 II 型 IFN 受体缺陷的小鼠，在感染后 5 天检测小鼠的神经组织。通过免疫组织化学检测体内 ER 应激的关键标志物磷酸化 IRE1、XBP1 和 ATF6 的表达。此外，通过免疫荧光检测 XBP1s 和 ATF6n 的核定位。此外，选择两种代表性的神经细胞，神经母细胞瘤细胞系（SK-N-SH）和星形细胞瘤细胞系（CCF-STTG1），在体外验证 ER 应激。通过 Western blot 分析 BIP、磷酸化 elF2α、磷酸化 IRE1 和 ATF6 的表达，并通过共聚焦免疫荧光显微镜检测 XBP1s 的核定位。还通过 RT-qPCR 定量体外和体内 UPR 下游基因的 mRNA 水平。
ZIKV 感染显著上调了体外和体内 ER 应激标志物的表达。磷酸化 IRE1 和 XBP1 的表达在小脑和中脑显著增加，而 ATF6 的表达在中脑显著增加。ATF6n 和 XBP1s 易位到细胞核。体外 BIP、ATF6、磷酸化 elF2α 和剪接 xbp1 的水平也显著增加。此外，还检测了 UPR 的下游基因，以研究 ZIKV 感染期间 UPR 的调节模型。体内 atf4、gadd34、chop 和 edem-1 的转录水平以及体外 gadd34 和 chop 的转录水平显著增加。
本研究结果表明，ZIKV 感染激活了神经细胞中的内质网应激。这些结果为进一步研究 ZIKV 感染引起的神经发病机制提供了线索。
Zotero 插件
