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Paragraph: In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6)  (33, 34) . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP (DDIT3) (35) . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ (p-eIF2␣). Tunicamycin, a glycosylation inhibitor and inducer of UPR (36) , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins (data not shown). VEEV-infected but not mock-infected or UV-inactivated VEEV (UV-VEEV)-infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi (Fig. 6A) . No change in protein expression was observed at 4 hpi (data not shown). Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells (Fig. 6C to E). While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection ( Fig. 5B and D). These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction (37) . As eIF2␣ can be phosphorylated by multiple kinases (PERK, protein kinase double-stranded RNA dependent [PKR], general control nonderepressible-2 [GCN2], and hemeregulated inhibitor [HRI]) (38) , the PERK inhibitor (PERKi) GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation (Fig. 6B) . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors (39, 40) . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus (41) (42) (43) . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner (44) . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection ( Fig. 4 and 5) , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication (likely due to cellular membrane disruption following entry) (41), a UV-inactivated virus control (UV-VEEV) was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells (Fig. 7A; compare lanes 3, 6, and 9 ). The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production (Fig. 1B) . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence (EBS) [GCG(G/T)GGCG] (40, 45) . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells (Fig. 7B) . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction (Fig. 7C) , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis (46) , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 (Fig. 8A ). In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability (ATP production) and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined (Fig. 8B) . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 (Fig. 8C) . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 (Fig. 8D) . EGR1 has been shown to negatively regulate the transcription of BIRC5 (survivin), an inhibitor of apoptosis (IAP) family member (47) . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively (see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864). WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs (Fig. 8E) . Ex-pression of the gene for the X-linked inhibitor of apoptosis (XIAP), another IAP family member, was not significantly differentially altered after infection (data not shown). Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs (0.5 and 5), and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi (Fig. 9A) . A lack of EGR1 expression was confirmed by Western blotting (Fig. 9B) . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression (Fig. 9D ) without any significant effect on viral replication (Fig. 9C) . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response (1, 48) . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways (1, 48) . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ (IL-1␤), IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha (TNF-␣), have all been shown to play a role in VEEV pathogenesis (49) (50) (51) (52) (53) . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production (54, 55) . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication (34) . Studies with hepatitis E virus (HEV) have demonstrated that expression of HEV capsid protein open reading frame 2 (ORF2) activates the expression of CHOP and ATF4 (56) . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements (AARE) through interaction with ATF4 (56) . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. (E) EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected (MOI, 5). RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 (survivin). The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle (C T ) method. *, P Ͻ 0.005 (comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells). inhibition of protein synthesis (33, 34) . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP (33, 34) . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins (57) . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 (Bcl-2). CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein (GADD34) in a self-regulating feedback look (33, 34) . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones (1, 58) . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins (nsPs) of Chikungunya virus (CHIKV) have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP (59) .

Question: What indicators does the UPR pathway use to regulate protein folding and secretion in the cell?
inositolrequiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6)