Murine Fibroblast Lipidomics and the Role of Raftassociated Type I Interferon Receptor Upon WNV Infection
Abstract
West Nile virus (WNV) is a mosquito-borne pathogen that causes neuroinvasive diseases in birds, horses, and humans. As a member of RNA viruses from the Flaviviridae family, viral replication in host cell cytoplasm markedly induces the alteration in the intracellular membranes and drastically modifies cellular lipid metabolism. In this study, whole cell lipidomics of infected mouse embryonic fibroblasts (MEFs) were investigated. We reported the drastic induction of total glycerophospholipids, sphingolipids, sterols, and fatty acyls upon WNV infection. Consistent with the suppression of viral titers, pretreatment with IFNβ increased the levels of specific sphingomyelin (SM) molecular species that are mostly abundant in raft microdomains. However, this specific induction of SM molecules was prominent only in wild-type cells containing the competent interferon reguratory factors (IRFs). The defect in IRF 3/5/7 triple-knockout MEFs correlated with the failure of inducing major SM molecules. To evaluate this effect, we manipulated SM contents in mouse fibroblasts with a xanthate compound named D609. Treatment with D609 was able to suppress total SM levels by 19.50% and 43.73% in wild-type and Irf3-/-Irf5-/-Irf7-/- triple-knockout MEFs, respectively. This compound effectively attenuated viral titers in both the highly pathogenic WNV and non-pathogenic Kunjin virus. In addition, viral attenuation was more significant when D609 was used in conjunction with IFN? pretreatment. Since SM is the main component of lipid rafts and these microdomains are involved with signal transduction processes, the association between raft-associated SM and type I IFN response was examined. The expression of type I interferon receptor (IFNAR1) and STAT1 in detergent-resistant and detergent-sensitive membrane fractions was analyzed in the infected MEFs under different treatment conditions. Our results revealed the predominant raft-localization of IFNAR1 following WNV infection only in cells with the fully functional IRFs. Due to the inhibitory effect of D609 on major SM molecules, treatment with this compound efficiently decreased the levels of IFNAR1 and the downstream signaling protein STAT1 in mouse fibroblasts. This information emphasized the requirement of raft-associated SM not only in structural function but also in the initiation of anti-viral type I IFN signal transduction initiated at the membrane receptor level. Accordingly, SM manipulation, in addition to type I IFN treatment, is suggested as a promising strategy in the control of WNV-related disease.
Degree
Ph.D.
Advisors
Kuhn, Purdue University.
Subject Area
Virology
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