The mosquito-borne flavivirus, Zika virus (ZIKV), was first isolated in 1947 from a rhesus monkey in the East African country of Uganda [
1]. ZIKV infection in humans was first reported in 1952 and only 14 cases of sporadic infection have been previously documented until an outbreak was reported by physicians on Yap Island in 2007 [
2‐
4]. The unprecedented epidemics of ZIKV among the Americas in 2015 have raised alarm due to its rapid transmission and association with microcephaly in newborns and serious neurological complications in adults, such as Guillain–Barre syndrome [
4‐
7]. The World Health Organization declared a public health emergency to maintain international concern regarding the virus [
8]. The relationship between ZIKV infection and neurodevelopment abnormalities has attracted more and more attention [
5,
7,
9,
10]. ZIKV, like other members of the
Flavivirus genus, is a positive (+) single-strand RNA virus. An approximately 10.7 kb genome of ZIKV encodes a single polyprotein precursor that is posttranslationally cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral and host proteases [
11‐
13]. Nonstructural proteins induce the formation of a membranous network with ER where viral replication occurs [
14]. Immature virions assemble within the ER, where viral RNA is complexed with the C protein and packaged into an ER-derived lipid bilayer containing heterodimers of prM and E proteins [
15]. Immature virions then bud into the ER lumen and are transported through the
trans-Golgi network. In the Golgi apparatus, glycan modification and structural cleavage (furin-mediated cleavage of the prM to M) are accomplished, and mature infectious virions are secreted into extracellular space via exocytosis [
14,
15].
The ER is an important location for posttranslational modification, folding, and oligomerization of secretory and cell surface proteins. It is also a main intercellular signal-transducing organelle for responding to environmental changes. Many viruses depend on the extensive membranous network of the ER for their translation, replication, and packaging [
16]. The
Flaviviridae family, including the dengue virus (DENV) [
16,
17], West Nile virus (WNV) [
18,
19], yellow fever virus (YFV), hepatitis C virus (HCV) [
20], and Japanese encephalitis virus (JEV) [
17], depend on the ER for their life cycles and are called endoplasmic reticulum tropic (ER-tropic) viruses [
16]. Infection by an ER-tropic virus disrupts the normal ER function, and then ER stress is induced. To alleviate ER stress, the UPR is activated and mainly functions in translational attenuation, protein folding, protein degradation, and cellular apoptosis [
21,
22]. PKR-like ER kinase (PERK), transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) are the sensors of the UPR pathway. In unstressed cells, the ER chaperone immunoglobulin heavy-chain-binding protein (BIP) binds to the ER luminal domain of the three sensors. Under the condition of ER stress, however, BIP is dissociated from the three sensors and preferentially binds to misfolded and unfolded proteins. Then, the three response pathways are activated to deal with different ER stress states in a time-dependent manner [
16]. Phosphorylated PERK phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylated eIF2α (phospho-eIF2α) forms a complex with guanine nucleotide exchange factor (eIF2B) and inhibits catalysis of GDP-GTP exchange, thereby leading to the translation attenuation and contradictory expression by activating transcription factor 4 (ATF4). Activated ATF4 upregulates a series of genes related to encoding metabolism and redox regulation and helps cells recover from ER stress. Growth arrest and DNA damage-inducible protein (GADD34), which is regulated by ATF4, interacts with protein phosphatase 1 (PP1) to dephosphorylate eIF2α, thereby acting as a negative feedback loop to restore protein translation [
23]. PERK-mediated eIF2α phosphorylation is triggered in early DENV-2 infection and suppressed in mid and late DENV-2 infection because the inhibition of eIF2α phosphorylation is necessary for viral protein synthesis [
16]. The RNase activity of phosphorylated IRE1 (phospho-IRE1) has only one substrate, X-box binding protein 1 (XBP1), and removes a 26-nucleotide intron from unspliced
xbp1mRNA (
xbp1u) [
24,
25]. The spliced
xbp1mRNA (
xbp1s) results in a frameshift mutation and encodes spliced XBP1 (XBP1s), a transcription factor that binds to the sequences of ER stress response elements (ERSEs) and UPR elements (UPREs), and upregulates the transcription of ER-association degradation (ERAD) proteins, proapoptotic proteins, and some ER chaperones [
26]. JEV infection activates the IRE1-XBP1 pathway and has a beneficial effect on the activation of the regulated IRE1-dependent decay (RIDD) pathway during the viral life cycle [
17,
27]. Cleaved ATF6 (ATF6n) also can bind to the ERSE and UPRE sequences and upregulate transcription chaperones, foldases, and lipid synthesis genes [
26]. ATF6 is required for efficient WNV replication by maintaining cell viability and modulating the innate immune responses [
28]. In brief, ER is an important location in viral life cycle, and UPR is closely associated with the regulation of viral replication. ZIKV is a flavivirus that its life cycle also closely depends on the extensive membranous network of the ER [
29]. However, whether ZIKV activates and benefits from ER stress to regulate viral replication needs to be investigated.
Many neurological diseases are linked to abnormal protein accumulation in the ER and activation of UPR to deal with ER stress [
30]. Khajavi and Lupski reported that the UPR is responsible for the demyelination in peripheral neuropathy [
31]. Yang and Paschen proposed that the UPR dysfunction after brain ischemia contributes to neuronal death [
32]. Several studies found a clear activation of the UPR in toxicological models of Parkinson’s disease. ATF6, XBP1, and pro-apoptotic transcription factor CCAT/enhancer-binding protein (CHOP) play functional roles in controlling dopaminergic neuron survival [
33]. In addition, whether the UPR plays an important role in the neuropathogenesis caused by ZIKV infection has yet to be studied in animal models or cellular level. In the present study, we found that ZIKV infection activated ER stress both in vitro and in vivo, showing that the expression of ER stress markers, namely, BIP, cleaved ATF6, phospho-IRE1, and phospho-eIF2α, significantly increased. The regulating model of UPR was developed, with ZIKV infection activating the IRE1-XBP1 pathway to regulate cellular apoptosis mediated by CHOP. This study could serve as a reference for elucidating ZIKV neuropathogenesis.