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West Nile virus and Zika virus infections induce aggresome formation in human neural progenitor and A549 cells

ABSTRACT: ZIKA viral replication vesicles (VRVs) were previously observed to cluster asymmetrically on one side of the nucleus in infected Huh7, A549 and human neural progenitor cells (h-NPCs). West Nile virus (WNV) infections in these cells also induce asymmetrically clustered VRVs. The sequential cell responses leading to asymmetric clustering of the WNV and ZIKV VRVs were investigated. Both infections activated the cellular unfolded protein response (UPR) as indicated by increased expression of HSPA5 (GRP78), the master regulator of endoplasmic reticular stress, and activation of the PERK branch of the UPR (p-PERK–p-eIF2α–GADD34). Increased microtubule (MT) acetylation was also detected. As the infection progressed, the level of ubiquitinated proteins increased and GRP78, HDAC6, ubiquitinated proteins and VRVs clustered at the cellular microtubule organizing center (MTOC) and became surrounded by a vimentin “cage.” The components involved in the formation of asymmetric virus replication complexes are characteristic of aggresome formation, an additional cellular response that copes with excessive misfolded proteins when the UPR is overwhelmed. During aggresome formation, HDAC6 links polyubiquitinated proteins to the dynein motor complex for transport along MTs to the MTOC where they aggregate and are surrounded by a vimentin cage. HDAC6 knockdown prevented the asymmetric clustering of the viral replication complexes and resulted in activation of the p-PERK–p-eIF2α–CHOP apoptotic pathway. The threshold for triggering aggresome formation varied with the infecting orthoflavivirus and cell type. The formation of aggresome-like, asymmetric VRVs in A549 cells and h-NPCs infected with WNV or ZIKV prolonged cell survival and increased the viral yield.

ORGANISM(S): Homo sapiens

PROVIDER: GSE310262 | GEO | 2026/03/30

REPOSITORIES: GEO

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			Action	DRS
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	GSE310262_Table_S1_DEGs_16h.xlsx	Xlsx
	GSE310262_Table_S2_DEGs_32h.xlsx	Xlsx

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Project description:Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated mice (cGrp78f/f) with lung epithelial cell-specific knockout (KO) of Grp78, a gene encoding the ER chaperone 78-kDa glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78f/f pups died immediately after birth, likely due to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-Î² (TGF-Î²)/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress and TGF-Î²/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone taursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78f/f lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress and suggest the UPR as a potential therapeutic target in BPD. Whole-genome expression profiling was performed using MouseRef-8 v2.0 Expression BeadChips (Illumina) on RNA isolated from lungs of four Grp78f/f and three cGrp78f/f mice at E18.

Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

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West Nile virus and Zika virus infections induce aggresome formation in human neural progenitor and A549 cells

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