The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis

Abstract

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. IMPORTANCEWest 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.

Host sphingomyelin increases West Nile virus infection in vivo

“…Viruses can take advantage of these properties to develop specialized membrane sites for RNA replication and particle biogenesis ( 18 ). Lipidomic analyses have shown an increase in the content of both ceramide and SM in fl avivirusinfected cells ( 8,12 ), and ceramide has specifi cally been associated with WNV replication and viral particle biogenesis ( 8,9 ). Regarding SM, it is enriched in membranes from the replication complex of viruses phylogenetically related to WNV, such as dengue virus and hepatitis C virus ( 12,19 ).…”

“…Regarding SM, it is enriched in membranes from the replication complex of viruses phylogenetically related to WNV, such as dengue virus and hepatitis C virus ( 12,19 ). For WNV, an enrichment of SM in the viral envelope relative to total cellular membranes has been described ( 8 ). Despite this observation, the contribution of SM, the most abundant complex sphingolipid in mammalian cells ( 15 ), has not been addressed in detail in WNV infection.…”

“…A possible explanation for this discrepancy is that treatment with SMS inhibitors increased the production of noninfectious genome-containing WNV particles. Considering that SM could play a structural role in WNV particles ( 8 ), the expected reduction on SM content due to SMS inhibition could result in a reduced availability of SM for viral envelopment, thus causing a decrease in the infectious viral particles produced. Similar effects have been observed in other viral models using other drugs that also block lipid synthesis (48)(49)(50).…”

“…In a parallel manner to that described for other fl aviviruses, both RNA replication and acquisition of lipid envelope are associated to specialized membranous structures derived from the endoplasmic reticulum (ER) ( 3-6 ). To generate this adequate environment for viral multiplication, WNV promotes the synthesis and accumulation of specifi c lipids (fatty acids, cholesterol, glycerophospholipids, and sphingolipids) within infected cells ( 5,(7)(8)(9). This selective manipulation of host cell lipid metabolism is not a unique feature of WNV infection, as it is also observed in cells infected with other fl aviviruses ( 10-12 ).…”

Usutu virus: current knowledge and future perspectives

“…40,41 Likewise, the involvement of neutral sphingomyelinase in USUV infection has unveiled a connection between sphingomyelin metabolism and USUV biogenesis. 42,43 Moreover, infection with USUV triggered an autophagic response in the host cell ( Figure 4). 44 Autophagy is an important cellular pathway that plays important roles in viral infections and pathogenesis, and the upregulation of the autophagic pathway has been well documented for various flaviviruses, 45 although the involvement of autophagy in WNV infection is still controversial.…”