**3. Flavivirus Propagation and Sphingolipids**

Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV) are archetype flaviviruses transmitted by mosquitoes, mainly *Aedes aegypti* and *Aedes albopictus*. According to the world health organization, DENV is responsible for 50–100 million infections each year with mild complications resembling flu-like symptoms and major complications, including deadly dengue hemorrhagic fever. WNV causes flu-like symptoms, neuroinvasive disease, and death in many countries in the world. WNV was first introduced in the United States (US) in 1999 from infected Israeli birds imported into New York state. The virus has now spread to most states and is responsible for many deaths in birds, humans and horses. By contrast, ZIKV only emerged as a global health concern since 2016, due its association with neurological disorders, such as microcephaly in newborns and Guillain–Barré syndrome in adults [27,43]. Like hepatitis C virus (HCV), DENV, WNV, and ZIKV are enveloped viruses. Unlike HCV, the positive-stranded RNA genome of these flaviviruses encodes a slightly different set of structural proteins (Core, E, and prM) (Figure 3) required for virus particle formation, and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) involved in viral genome replication, packaging, and pathogenesis [44–46]. No effective vaccine or specific antiviral treatments are currently available for DENV, WNV, or ZIKV infection.

**Figure 3.** Organization of a flavivirus genome. The genome of flaviviruses, such as dengue and Zika, is ~11 kb in size and encodes a single, large polyprotein, which is proteolytically processed into three structural proteins and seven nonstructural proteins. The 5' end of the genome contains a cap structure critical for the initiation of translation. RNA structures present in the 5' and 3' untranslated regions (UTR) are critical for capping and genome replication.

#### *3.1. DENV Propagation and Sphingolipids*

There is evidence that sphingolipids and glycosphingolipids are also required for the replication of some flaviviruses. For example, DENV has been reported to upregulate the expression of sphingolipids (ceramide and sphingomyelin) in mosquito cells and cause an accumulation of these lipids in a membrane fraction enriched in the viral replication complex [30]. That study did not directly address the role of sphingolipids in DENV replication. In a different study, Wang et al. [47] exploited mouse melanoma WT cells (B16) and a mutant counterpart (GM95) to demonstrate that the glycosphingolipid GM3 is required for DENV genome replication. The authors found higher levels of GM3 in DENV-infected cells and relocalization of GM3 to sites where DENV replicates its genome. Importantly, the authors found that inhibition of GM3 synthesis with soyasaponin I increases the survival rate of DENV-infected mice [47]. While the exact role of GM3 in DENV genome replication is currently unknown, these in vivo findings raise the prospect that pharmacological inhibitors targeting GM3 synthesis can serve as a foundation for new antiviral therapy.

#### *3.2. WNV Propagation and Sphingolipids*

3.2.1. Sphingolipids and WNV Entry and Genome Replication

The role of sphingomyelin in WNV replication is well documented. A study by Martin-Acebes and colleagues [48] showed that WNV replicates at a much higher level in mice deficient in acid sphingomyelinase (unable to catabolize sphingomyelin), or cells derived from Niemann–Pick disease type A patients (NPA; accumulate sphingomyelin) relative to the their wild type controls. This suggested that sphingomyelin accumulation enhances WNV infectivity. Consistent with these findings, adding sphingomyelin to infected fibroblast cells markedly increased WNV infectivity. Further analysis showed that sphingomyelin colocalizes with WNV dsRNA at cytoplasmic foci, implying that sphingomyelin plays a role in the formation of the WNV replication platform [48]. Interestingly, pharmacological inhibitors of sphingomyelin synthesis (DS609 and SPK-601) markedly reduced the infectivity of WNV released from infected cells, but had little impact on the amount of released viral genome [48]. These findings imply that sphingomyelin is also required for WNV attachment, internalization, and/or virus–endosome fusion.
