West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and
meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific
lipids, such as
cholesterol and
fatty acids, has been documented. In this study, we evaluated the impact of
WNV infection on two important components of cellular membranes,
glycerophospholipids and
sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several
glycerophospholipids (
phosphatidylcholine,
plasmalogens, and
lysophospholipids) and
sphingolipids (
ceramide,
dihydroceramide, and
sphingomyelin) was noticed in WNV-infected cells, suggesting that these
lipids have functional roles during
WNV infection. Furthermore, the analysis of the
lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in
sphingolipids (
sphingomyelin) and showed reduced levels of
phosphatidylcholine, similar to
sphingolipid-enriched
lipid microdomains. Inhibition of neutral
sphingomyelinase (which catalyzes the hydrolysis of
sphingomyelin into
ceramide) by either pharmacological approaches or
small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of
sphingomyelin-to-
ceramide conversion in flavivirus budding and confirming the importance of
sphingolipids in the biogenesis of WNV. Importance: West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific
vaccine or
therapy against this pathogen licensed for human use. Since the multiplication of this virus is associated with rearrangements of host cell membranes, we analyzed the effect of
WNV infection on different cellular
lipids that constitute important membrane components. The levels of multiple
lipid species were increased in infected cells, pointing to the induction of major alterations of cellular lipid metabolism by
WNV infection. Interestingly, certain
sphingolipids, which were increased in infected cells, were also enriched in the
lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of
sphingolipids in the production of WNV by means of functional analyses. This study provides new insight into the formation of flavivirus infectious particles and the involvement of
sphingolipids in the WNV life cycle.