Zika virus (ZIKV) is an emergent member of the Flaviviridae family which causes severe congenital defects and other major sequelae, but the cellular processes that support ZIKV replication are incompletely understood. Related flaviviruses use the endoplasmic reticulum (ER) as a membranous platform for viral replication and induce ER stress during infection. Our data suggest that ZIKV activates IRE1α, a component of the cellular response to ER stress. IRE1α is an ER-resident transmembrane protein that possesses a cytosolic RNase domain. Upon activation, IRE1α initiates nonconventional cytoplasmic splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor, which upregulates ER-related targets. We find that ZIKV infection induces XBP1 mRNA splicing and induction of XBP1 target genes. Small molecule inhibitors of IRE1α, including those specific for the nuclease function, prevent ZIKV-induced cytotoxicity, as does genetic disruption of IRE1α. Optimal ZIKV RNA replication requires both IRE1α and XBP1. Spliced XBP1 has been described to cause ER expansion and remodeling and we find that ER redistribution during ZIKV infection requires IRE1α nuclease activity. Finally, we demonstrate that inducible genetic disruption of IRE1α and XBP1 impairs ZIKV replication in a mouse model of infection. Together, our data indicate that the ER stress response component IRE1α promotes ZIKV infection via XBP1 and may represent a potential therapeutic target.proteins: protein kinase receptor-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 α (IRE1α). ER stress causes IRE1α to undergo oligomerization and autophosphorylation, which activates its cytosolic RNase domain to initiate nonconventional splicing of XBP1 mRNA. Spliced XBP1 is a specific product of activated IRE1α and encodes a transcription factor that upregulates targets that are involved in ER function [11]. IRE1α also targets other specific RNAs, leading to their degradation in a process termed regulated IRE1-dependent decay (RIDD) [12,13].The role of IRE1α in infection appears to vary for different members of the Flaviviridae family. Hepatitis C virus (HCV) activates IRE1α [14] to promote viral replication [15] independently of XBP1 by preventing apoptotic death of infected cells [16]. Dengue (DENV) and Japanese encephalitis viruses (JEV) also benefit from IRE1α via an XBP1-independent mechanism [17-20], whereas West Nile virus (WNV) replication is unaffected by either IRE1α [21,22] or XBP1 [23]. Conflicting results have been obtained for tick-borne encephalitis virus, with IRE1α nuclease inhibition either limiting viral replication [24] or having no effect [22]. ZIKV activates IRE1α, as demonstrated by the presence of spliced XBP1 in ZIKV-infected cultured cells and brain tissue from ZIKV-infected embryonic mice [25][26][27]. In this study, we examined the role of IRE1α in ZIKV infection and found that IRE1α promotes ZIKV replication via XBP1 in cultured cells. We further found that genetic disruption of IRE1α and XBP1 lim...

The cellular processes that support human coronavirus replication and contribute to the pathogenesis of severe disease remain incompletely understood. Many viruses, including coronaviruses, cause endoplasmic reticulum (ER) stress during infection. IRE1α is a component of the cellular response to ER stress that initiates non-conventional splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor that induces the expression of ER-related targets. Activation of the IRE1α–XBP1 pathway occurs in association with risk factors for severe human coronavirus infection. In this study, we found that the human coronaviruses HCoV-OC43 (human coronavirus OC43) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) both robustly activate the IRE1α–XBP1 branch of the unfolded protein response in cultured cells. Using IRE1α nuclease inhibitors and genetic knockdown of IRE1α and XBP1, we found that these host factors are required for optimal replication of both viruses. Our data suggest that IRE1α supports infection downstream of initial viral attachment and entry. In addition, we found that ER stress–inducing conditions are sufficient to enhance human coronavirus replication. Furthermore, we found markedly increased XBP1 in circulation in human patients with severe coronavirus disease 2019 (COVID-19). Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection. IMPORTANCE There is a critical need to understand the cellular processes co-opted during human coronavirus replication, with an emphasis on identifying mechanisms underlying severe disease and potential therapeutic targets. Here, we demonstrate that the host proteins IRE1α and XBP1 are required for robust infection by the human coronaviruses, SARS-CoV-2 and HCoV-OC43. IRE1α and XBP1 participate in the cellular response to ER stress and are activated during conditions that predispose to severe COVID-19. We found enhanced viral replication with exogenous IRE1α activation, and evidence that this pathway is activated in humans during severe COVID-19. Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.

Background: The number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. Results: We found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. Conclusion: Our findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.