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.

Enterovirus A71 (EVA71) is a human enterovirus belonging to the Picornaviridae family and mostly causes hand-foot-and-mouth disease in infants. Viperin is an important interferon-stimulated gene with a broad antiviral activity against various viruses. However, the effect of viperin on human enteroviruses and the interaction mechanism between EVA71 and viperin remains elusive. Here, we confirmed the EVA71-induced expression of viperin in a mouse model and cell lines and showed that viperin upregulation by EVA71 infection occurred on both the mRNA and protein level. Viperin knockdown and overexpression in EVA71-infected cells indicated that this protein can markedly inhibit EVA71 infection. Interestingly, immunofluorescent confocal microscopy and co-immunoprecipitation assays indicated that viperin interacts and colocalizes with the EVA71 protein 2C in the endoplasmic reticulum. Furthermore, amino acids 50–60 in the N-terminal domain of viperin were the key residues responsible for viperin interaction with 2C. More importantly, the N-terminal domain of viperin was found responsible for inhibiting EVA71 replication. Our findings can potentially aid future research on the prevention and treatment of nervous system damage caused by EVA71 and may provide a potential target for antiviral therapy.

Enterovirus 71 (EV71) is the major causative pathogen of human hand, foot, and mouth disease (hHFMD) and has evolved to use various cellular receptors for infection. However, the relationship between receptor preference and EV71 virulence has not been fully revealed. By using reverse genetics, we identified that a single E98K mutation in VP1 is responsible for rapid viral replication in vitro. The E98K mutation enhanced binding of EV71-GZCII to cells in a heparan sulfate (HS)-dependent manner, and it attenuated the virulence of EV71-GZCII in BALB/c mice, indicating that the HS-binding property is negatively associated with viral virulence. HS is widely expressed in vascular endothelial cells in different mouse tissues, and weak colocalization of HS with scavenger receptor B2 (SCARB2) was detected. The cGZCII-98K virus bound more efficiently to mouse tissue homogenates, and the cGZCII-98K virus titers in mouse tissues and blood were much lower than the cGZCII virus titers. Together, these findings suggest that the enhanced adsorption of the cGZCII-98K virus, which likely occurs through HS, is unable to support the efficient replication of EV71 in vivo. Our study confirmed the role of HS-binding sites in EV71 infection and highlighted the importance of the HS receptor in EV71 pathogenesis.