Citation: Lehman, C.W.; Smith, A.; Kelly, J.; Jacobs, J.L.; Dinman, J.D.; Kehn-Hall, K. EGR1 Upregulation during Encephalitic Viral Infections Contributes to Inflammation and Cell Death. Viruses 2022, 14, 1210. https://doi.org/10.3390/v14061210 Academic Editor: Patricia M. Legler Received: 20 April 2022 Accepted: 30 May 2022 Published: 2 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article EGR1 Upregulation during Encephalitic Viral Infections Contributes to Inflammation and Cell Death Caitlin W. Lehman 1,2 , Amy Smith 1,2 , Jamie Kelly 3 , Jonathan L. Jacobs 4 , Jonathan D. Dinman 3 and Kylene Kehn-Hall 1,2, * 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA; woodsonc@vt.edu (C.W.L.); amysmith104@gmail.com (A.S.) 2 Center for Emerging, Zoonotic, and Arthropod-Borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA 3 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA; jkelly22@umd.edu (J.K.); dinman@umd.edu (J.D.D.) 4 American Type Culture Collection, Manassas, VA 20110, USA; jjacobs@atcc.org * Correspondence: kkehnhall@vt.edu Abstract: Early growth response 1 (EGR1) is an immediate early gene and transcription factor previously found to be significantly upregulated in human astrocytoma cells infected with Venezuelan equine encephalitis virus (VEEV). The loss of EGR1 resulted in decreased cell death but had no significant impact on viral replication. Here, we extend these studies to determine the impacts of EGR1 on gene expression following viral infection. Inflammatory genes CXCL3, CXCL8, CXCL10, TNF, and PTGS2 were upregulated in VEEV-infected cells, which was partially dependent on EGR1. Additionally, transcription factors, including EGR1 itself, as well as ATF3, FOS, JUN, KLF4, EGR2, and EGR4 were found to be partially transcriptionally dependent on EGR1. We also examined the role of EGR1 and the changes in gene expression in response to infection with other alphaviruses, including eastern equine encephalitis virus (EEEV), Sindbis virus (SINV), and chikungunya virus (CHIKV), as well as Zika virus (ZIKV) and Rift Valley fever virus (RVFV), members of the Flaviviridae and Phenuiviridae families, respectively. EGR1 was significantly upregulated to varying degrees in EEEV-, CHIKV-, RVFV-, SINV-, and ZIKV-infected astrocytoma cells. Genes that were identified as being partially transcriptionally dependent on EGR1 in infected cells included ATF3 (EEEV, CHIKV, ZIKV), JUN (EEEV), KLF4 (SINV, ZIKV, RVFV), CXCL3 (EEEV, CHIKV, ZIKV), CXCL8 (EEEV, CHIKV, ZIKV, RVFV), CXCL10 (EEEV, RVFV), TNF-α (EEEV, ZIKV, RVFV), and PTGS2 (EEEV, CHIKV, ZIKV). Additionally, inhibition of the inflammatory gene PTGS2 with Celecoxib, a small molecule inhibitor, rescued astrocytoma cells from VEEV-induced cell death but had no impact on viral titers. Collectively, these results suggest that EGR1 induction following viral infection stimulates multiple inflammatory mediators. Managing inflammation and cell death in response to viral infection is of utmost importance, especially during VEEV infection where survivors are at-risk for neurological sequalae. Keywords: Venezuelan equine encephalitis; astrocytes; EGR1; inflammation; alphaviruses 1. Introduction Alphaviruses belong to the viral family Togaviridae and are further subdivided into either Old World or New World alphaviruses based on their genetic sequence and the type of clinical disease they cause [1]. Old World alphaviruses, including Sindbis virus (SINV) and chikungunya virus (CHIKV), induce arthritic disease and are primarily endemic to Africa, Asia, Europe, Australia, and Central and South America [2]. Although not as common, both CHIKV and SINV can also cause neurological disease [3]. Meanwhile, New World alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern Viruses 2022, 14, 1210. https://doi.org/10.3390/v14061210 https://www.mdpi.com/journal/viruses