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Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

Survival curves based on clinical information obtained from GSE7696 and GSE16011 for Endothelin receptor type B (ETBR) mRNA expression in GBM. GSE7696 and GSE16011 cohort shows that over-median expression of ETBR has lower survival rate at 3 years than 5 years ( http://watson.compbio.iupui.edu/ ). Figure S2. ETBR mRNA expression in GSE42656 (A) and GSE50161 (B). No statistically significant difference was observed for ETBR expression in normal brain tissue compared to that of GBM patients, mirroring heterogeneity of disease. Figure S3. Overexpression of ETBR mRNA in different cancers of the TCGA cancer cohort is shown using The Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle ). Numbers in parentheses indicate sample size. Figure S4. The ETBR-interacting proteins (up to second neighbor) were searched in different cancer signature genes as described [22] A). The number of ETBR-interacting proteins found to be signature genes in various cancer cohorts were shown. B) The association between cancer types and ETBR-interacting proteins suggests a possible role of ETBR in GBM and other cancers. Cancer types are shown with degree-based node shape and surrounding red circles, whereas ETBR-interacting proteins are shown with degree based nodes (orange). Figure S5. ETBR expression in different subtypes of GBM (A) and its correlation with survival (B) according to molecular classification [21]. Higher expression of ETBR in both ‘Classical’ and ‘Neural’ subtype tended to be correlated with poor overall survival. Figure S6. Relative expression of ETBR in various primary GBM cells, breast cancer lines (MCF-7, SKBR3 and MDA-MB-231), fibroblast (MRC-5), endothelial cells (HUVEC) and epithelial cells (RPE) as normalized to MRC-5. (DOC 2199 kb)

Survival curves based on clinical information obtained from GSE7696 and GSE16011 for Endothelin receptor type B (ETBR) mRNA expression in GBM. GSE7696 and GSE16011 cohort shows that over-median expression of ETBR has lower survival rate at 3 years than 5 years ( http://watson.compbio.iupui.edu/ ). Figure S2. ETBR mRNA expression in GSE42656 (A) and GSE50161 (B). No statistically significant difference was observed for ETBR expression in normal brain tissue compared to that of GBM patients, mirroring heterogeneity of disease. Figure S3. Overexpression of ETBR mRNA in different cancers of the TCGA cancer cohort is shown using The Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle ). Numbers in parentheses indicate sample size. Figure S4. The ETBR-interacting proteins (up to second neighbor) were searched in different cancer signature genes as described [22] A). The number of ETBR-interacting proteins found to be signature genes in various cancer cohorts were shown. B) The association between cancer types and ETBR-interacting proteins suggests a possible role of ETBR in GBM and other cancers. Cancer types are shown with degree-based node shape and surrounding red circles, whereas ETBR-interacting proteins are shown with degree based nodes (orange). Figure S5. ETBR expression in different subtypes of GBM (A) and its correlation with survival (B) according to molecular classification [21]. Higher expression of ETBR in both ‘Classical’ and ‘Neural’ subtype tended to be correlated with poor overall survival. Figure S6. Relative expression of ETBR in various primary GBM cells, breast cancer lines (MCF-7, SKBR3 and MDA-MB-231), fibroblast (MRC-5), endothelial cells (HUVEC) and epithelial cells (RPE) as normalized to MRC-5. (DOC 2199 kb)

Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo., Author Summary The suppression of the production rather than the blockage of action of the potent inflammatory mediator TNFα is a particular hallmark of anti-TNFα mechanisms associated with microbial and parasitic infections. Whether this mode of counter-regulation is an important feature of infection by viruses is not clear. Also, it remains to be determined whether a specific pathogen gene in the context of an infection in vivo is capable of modulating levels of TNFα production. In this study we disclose a virus-mediated moderation of TNFα production, dependent on the ie1 gene of murine cytomegalovirus (MCMV). The ie1 gene product IE1 is a well-characterized nuclear protein capable of altering levels of host and viral gene expression although its biological role in the context of a natural infection is to date unknown. We provide evidence showing that ie1 is associated with a moderated pro-inflammatory cytokine response, in particular with TNFα production. Further, we show that the viral moderation of this cytokine is not only readily apparent in vitro but also in the natural host. The identification of a viral gene responsible for this mode of regulation in vivo may have therapeutic potential in the future in both anti-viral and anti-inflammatory strategies.