West Nile Virus Infection Activates the Unfolded Protein Response, Leading to CHOP Induction and Apoptosis

Abstract

West Nile virus (WNV)-mediated neuronal death is a hallmark of WNV meningitis and encephalitis.However, the mechanisms of WNV-induced neuronal damage are not well understood. We investigated WNV neuropathogenesis by using human neuroblastoma cells and primary rat hippocampal neurons. We observed that WNV activates multiple unfolded protein response (UPR) pathways, leading to transcriptional and translational induction of UPR target genes. We evaluated the role of the three major UPR pathways, namely, inositol-requiring enzyme 1-dependent splicing of X box binding protein 1 (XBP1) mRNA, activation of activating transcription factor 6 (ATF6), and protein kinase R-like endoplasmic reticulum (ER) kinase-dependent eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation, in WNVinfected cells. We show that XBP1 is nonessential or can be replaced by other UPR pathways in WNV replication. ATF6 was rapidly degraded by proteasomes, consistent with induction of ER stress by WNV. We further observed a transient phosphorylation of eIF2␣ and induction of the proapoptotic cyclic AMP response element-binding transcription factor homologous protein (CHOP). WNV-infected cells exhibited a number of apoptotic phenotypes, such as (i) induction of growth arrest and DNA damage-inducible gene 34, (ii) activation of caspase-3, and (iii) cleavage of poly(ADP-ribose) polymerase. The expression of WNV nonstructural proteins alone was sufficient to induce CHOP expression. Importantly, WNV grew to significantly higher viral titers in chop ؊/؊ mouse embryonic fibroblasts (MEFs) than in wild-type MEFs, suggesting that CHOP-dependent premature cell death represents a host defense mechanism to limit viral replication that might also be responsible for the widespread neuronal loss observed in WNV-infected neuronal tissue.

Dengue Virus Modulates the Unfolded Protein Response in a Time-dependent Manner

“…We find that activation of the IRE1-XBP1 pathway correlates with expression of CHOP and GADD34 and that at these time points we detect similar levels of eIF2␣-P in DENV-2-infected cells and time-matched mock infected controls. This contrasts with previous reports, where eIF2␣-P was detected late in DENV and WNV infection (18,19). These differences with respect to DENV infection might be due in part to the mock infected controls chosen for comparison.…”

“…The up-regulation of downstream genes that reduce ER stress, such as GRP78 (Figs. 4C and 5A), is consistent with previously published reports for bovine viral diarrhea virus (16), WNV (19), and Japanese encephalitis virus (32); however, contrary to these reports, we do not observe changes in expression levels of GRP94 or PDI proteins, both of which are also involved in maintaining ER homeostasis.…”

“…However, we did not observe any effect on DENV-2 growth kinetics in PKR Ϫ/Ϫ compared with PKR ϩ/ϩ cells, consistent with our previous observations (Fig. 6A and data not shown) (16,19).…”

“…Although several reports have demonstrated that flavivirus infection activates the UPR (18,19), the effect of genetically knocking out individual components of the UPR on DENV-2 production has not been addressed. We began by testing components that affect the ISR arm of the UPR.…”

“…Infection with Japanese encephalitis virus and DENV-2 was shown to induce the IRE-XBP1 pathway and a subset of XBP1-activated genes involved in the ER-associated degradation pathway, protecting cells from virus-induced cytopathic effects (17). In contrast, another group suggested that DENV induction of IRE1-XBP1 and ATF6 pathways contributed to dengue pathogenesis (18), whereas WNV induction of the UPR was shown to lead to CHOP-mediated apoptosis (19). Although these studies demonstrated that viral infection can lead to induction of the individual branches of the UPR and their downstream components, an integrated study of the role the UPR plays during the DENV life cycle and how DENV may modulate the UPR is still lacking.…”