Ebola Virus Inhibits Induction of Genes by Double-Stranded RNA in Endothelial Cells

Abstract

Fatal cases of filoviral infection are accompanied by a marked immunosuppression. Endothelial cells play a vital role in the host immune response through the expression of several immunomodulatory genes in addition to the expression of the antiviral genes, 2',5'-oligoadenylate synthetase [2'-5'(A)N], and the double-stranded RNA (dsRNA)-activated protein kinase (PKR). dsRNA, an intermediate generated during viral replication and gene transcription of many viruses, leads to the induction of immunomodulatory genes in endothelial cells. In this report, we show that induction of the major histocompatibility complex class I family of genes, 2'-5'(A)N, interleukin-6 (IL-6), PKR, interferon (IFN)-regulatory factor-1, and intercellular adhesion molecule-1 (ICAM-1) by dsRNA in human umbilical vein endothelial cells is suppressed by infection with the filovirus Ebola-Zaire (EZ). In contrast, induction of IL-6 and ICAM-1 by IL-1 is intact in EZ-infected cells. Gel shift analysis demonstrates that dsRNA-induced protein binding to IFN-responsive elements is strongly suppressed by EZ-IFN, whereas NF-kappa B activation by dsRNA remains intact. We previously reported that IFN signaling is suppressed by EZ infection, and these data strongly suggest that elements shared between IFN and dsRNA signaling are being inhibited by EZ. Inhibition of IFN and dsRNA responsiveness could play a role in the immunosuppression seen in EZ infections and would play a role in the pathogenesis of disease caused by EZ.

IFN-α Sensitizes Human Umbilical Vein Endothelial Cells to Apoptosis Induced by Double-Stranded RNA

“…In this facility, HUVECs were prepared from umbilical veins, as previously described (19,27). Cells were cultured in medium 199 supplemented with 20% FCS (HyClone Laboratories, Logan, UT), 16 U/ml heparin (ESI Pharmaceuticals, Cherry Hill, NJ), 50 g/ml endothelial cell growth supplement (Biomedical Technologies, Stoughton, MA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen, Carlsbad, CA), and grown at 37°C on tissue culture plates coated with 0.4% gelatin (Sigma-Aldrich, St. Louis, MO).…”

“…When the hemorrhagic virus Ebola infects endothelial cells, key components of the innate antiviral machinery are blocked, leading to a loss of responsiveness to IFNs and to dsRNA (16,19). This disruption of the innate antiviral response has tremendous biological consequences because endothelial cells are heavily infected with Ebola, yet the cells are unable to respond to the virus or signal to the immune system to generate an inflammatory response, and the infection is usually fatal (20).…”

“…For example, dsRNA induces the expression of the cellular adhesion molecules ICAM-1, VCAM-1, and E-selectin (26,27), molecules that are important in adhesion and transendothelial migration of leukocytes that could help contain the viral infection (28,29). dsRNA also induces expression of MHC I (19), a protein critical for the presentation of foreign Ags to CTL, contributing to the acquired immune response (30,31). Endothelial cells also produce the cytokines IL-1␣, IL-1␤, IL-6, and type I IFN in response to dsRNA (26,32,33), thereby contributing to leukocyte maturation and alerting neighboring cells to the potential risk of viral infection.…”

“…Although endothelial cells are known to respond to dsRNA with multiple changes in gene expression and a reduction in protein synthesis (19,26,27), the role of apoptosis in the endothelial response to synthetic or viral dsRNA has not been reported. In this study, we demonstrate that multiple caspases are activated by dsRNA, and IFN-␣ priming dramatically accelerates their activation by dsRNA.…”

The Ebola virus VP35 protein functions as a type I IFN antagonist

“…After treatment with dsRNA, IFN-␣, or IFN-␥, little induction of IFN-stimulated genes was seen in infected cells compared with uninfected cells (24,25). However, this does not reflect a general inhibition of signal transduction; IL-1␤-induced signaling was not inhibited in Ebola virus-infected cells (24,25).…”