The arboviruses of the Flavivirus genus, Flaviviridae family members, as enveloped viruses, have a major and conserved E glycoprotein, which is composed of ninety dimers arranged with a quasi-icosahedral symmetry on the viral membrane (Nybakken et al., 2006; Li et al., 2008; Dai et al., 2016). This protein is highly conserved in the Flavivirus genus and is involved in viral attachment to the mosquito and mammalian host cells. The first step in viral infections is the binding and entry process. In this step, the viral particles have to bind to specific molecules on the cell surface such as receptors and coreceptors to enter into the host cell (Figure 1A). Since several of the attachment and receptor molecules described for viruses are present in lipid rafts (Takahashi and Suzuki, 2009), lipids and cholesterol-rich plasma membrane microdomains that are essential during entry of DENV (Lee et al., 2008; Puerta-Guardo et al., 2010; Soto-Acosta et al., 2013; García Cordero et al., 2014; Reyes-del Valle et al., 2014; Diwaker et al., 2015), and WNV (Medigeshi et al., 2008) (Figure 1A). The use of cholesterol-depleting drugs such as methyl-β-cyclodextrin (MβCD) or filipin, which binds to cellular cholesterol forming complexes and avoiding the formation of lipid rafts before viral entry. It has been demonstrated to induce an antiviral effect for DENV and WNV infection, which made it possible to confirm the importance of lipid rafts during entry for both viruses (Lee et al., 2008; Medigeshi et al., 2008; Rothwell et al., 2009). To the contrary, cholesterol and lipid rafts are not essential during the entry in the mosquito cell line C6/36 (Aedes albopictus) infected with DENV (Mosso et al., 2008) and WNV (Chu et al., 2006). Although insects cannot synthesize cholesterol de novo, they can take it to synthesize cell membranes and hormones (CLAYTON et al., 1962; CLAYTON, 1964; Krebs and Lan, 2003). Recently, cholesterol molecules were detected during the early stages of DENV replication in the middle intestine of Aedes aegypti mosquitoes (Chotiwan et al., 2018). However, the specific role of the cholesterol in this organ in mosquito-virus interaction remains unexplored.

The cholesterol dependence of DENV entry and post-entry steps have been observed in several mammalian cell lines (Lee et al., 2008; Soto-Acosta et al., 2013; Martínez-Gutierrez et al., 2014), it does not seem to be a general event, because it has been described that cholesterol is not required for Vero (green monkey epithelial kidney cell line) (Acosta et al., 2009; Carro and Damonte, 2013), HepG2 (Hepatocarcinoma cell line) and ECV304 cells entry (human endothelial cell line) (Rothwell et al., 2009). In addition, it has been described that the molecular tweezer CLR01, a small molecule that previously has been shown to inactivate some viruses through a selective interaction with the host-membrane-derived lipid bilayer of the viral envelope, inhibit EBOV (ebola virus) and ZIKV infection (Röcker et al., 2018).

Immediately after attachment and entry of the viral particles by clathrin-mediated endocytosis (Chu et al., 2006; Mosso et al., 2008; Acosta et al., 2009), at one and 48 h post-infection, an increase in cholesterol levels is observed in infected cells in mammalian cells (Soto-Acosta et al., 2013). This increment correlates with an increase in the presence of the low-density lipoprotein receptor (LDLr) on the surface of infected cells and with an augment in the cholesterol uptake (Soto-Acosta et al., 2013), indicating that cholesterol is essential during the first few hours of infection. On the other side, has been described that the structural protein C of DENV can interact with very low-density lipoproteins (VLDL) (Faustino et al., 2014). Faustino et al., using atomic force microscopy-based force spectroscopy, dynamic light scattering, nuclear magnetic resonance, and computational approach; demonstrated that dengue viral capsid proteins (C protein) bind to very low density lipoprotein (VLDL) surfaces (Faustino et al., 2014) (Figure 1A). This observation suggests the formation of lipoviroparticles (LVPs) in DENV infection. However, the presence of LVPs has not been observed during in vivo DENV infection, and the direct function of LVPs in DENV attachment or entry steps has not been analyzed (Reyes-del Valle et al., 2014). Besides, there is a report where apolipoprotein A-1 (Apo A-1), the main component of high-density lipoprotein (HDL), interact with DENV particles and facilitates viral entry through the scavenger receptor class B type I (SR-BI), the cell receptor for Apo A-I (Li et al., 2013) (Figure 1A). These observations provide evidence on the functional importance of lipoproteins and cholesterol uptake through cholesterol receptors during DENV infection. Moreover, the importance of the intracellular trafficking of cholesterol during the DENV entry has been demonstrated when this traffic is inhibited by the drug U18666A which mimic Niemann-Pick type C disease (hereditary lysosomal storage disease), causing the accumulation of cholesterol and the entrapment of DENV particles in late endosomes and lysosomes, reducing levels of viral genome released into the cytoplasm of treated cells (Poh et al., 2012).

As enveloped viruses, flaviviruses need to be uncoated to release the viral RNA into the cytoplasm (Figure 1B). Uncoating is induced by the low pH environment of the endosomes, where the viral proteins enter into a fusion-active state and initiate the merging of the viral envelope with the endosomal membrane, thereby releasing the viral RNA genome into the cytoplasm (Kaufmann and Rossmann, 2011; Smit et al., 2011). This process requires two steps, the fusion between the viral and endosomal membranes and the uncoating of the protective capsid shell (Rumlová and Ruml, 2018). To analyze the fusion event, the lipid composition of the viral membrane of different viruses has been characterized (Brügger et al., 2006; Kalvodova et al., 2009; Merz et al., 2011; Gerl et al., 2012; Reddy and Sansom, 2016). In the Flaviviridae family, an essential role of membrane virion-associated cholesterol has been demonstrated for all serotypes of DENV (Carro and Damonte, 2013). As it has been shown for HCV (Aizaki et al., 2008) (another member of the family Flaviviridae, but not an arbovirus), the presence of cholesterol in DENV virions was more crucial for infection (Carro and Damonte, 2013). In this regard, Carro and Damonte showed that after exposure of DENV particles to MβCD a loss of infectivity of the four serotypes, associated with a reduction in the cholesterol content of the virions was observed.

Moreover, the addition of exogenous water-soluble cholesterol or fetal bovine serum did not fully recover the infectivity of virions, except, when a simultaneous incubation with MβCD and serum cholesterol was performed (Carro and Damonte, 2013); this is consistent with others reports in which authors try to restore the cholesterol that has been depleted from viral particles (Sun and Whittaker, 2003; Lee et al., 2008; Desplanques et al., 2010).

Interestingly, when virions were incubated only with exogenous cholesterol, an inactivating effect on particle infectivity was observed. This effect is similar that the one observed by Lee et al., allowing to suggest that excess in cholesterol could induce an increase in envelope rigidity, which in turn would limit the ability of the viral membrane to fuse inhibiting viral entry (Lee et al., 2008). The MβCD removes not only cholesterol but also other components as phospholipids from the membranes. Therefore, the possibility that MβCD alters the composition of other phospholipids in the viral particle has to be considered (Zidovetzki and Levitan, 2007; Carro and Damonte, 2013). On the other hand, in an in vitro study, using a liposomal model system, it has been revealed that flaviviruses such as WNV can fuse with these receptor-free artificial lipid membranes, consisting of phosphatidylcholine and phosphatidylethanolamine at low pH, although with low efficiency. However, the addition of cholesterol to the target membranes has a strong promoting effect on the fusion capacity of WNV (Moesker et al., 2010). Other studies of virus-liposome co-flotation have indicated that cholesterol stimulates the interaction of glycoprotein E with lipid membranes (Stiasny et al., 2003; Umashankar et al., 2008), confirming the importance of cholesterol and specifically of the 3-hydroxyl group of cholesterol for this function. In contrast, the glycoprotein E of alphaviruses does not appear to interact directly with cholesterol in the target membrane (Umashankar et al., 2008). These observations suggest that cholesterol could be required to induce changes in the fluidity of the viral membrane or changes in the physicochemical properties of the membrane required during viral and endosomal membranes fusion (Smit et al., 2011). In an attempt to analyze the molecular composition of the flavivirus envelope, the composition of the lipid envelope of WNV virions was studied. Authors describe that the viral membrane is enriched in sphingolipids (sphingomyelin) and contains reduced levels of phosphatidylcholine (Martín-Acebes et al., 2014). Interestingly, by constructing a computational model of the DENV envelope, in which the known structure of the DENV membrane proteins was combined with a lipidic bilayer model. The authors found that despite the absence of cholesterol in the envelope, the virion presented a biophysical robustness that coincided with the level of cholesterol in the membrane of the influenza A virus (Reddy and Sansom, 2016). Thus, more studies have to be performed to determine the contribution of cholesterol to the biophysical properties of the viral particle. It is evident that the viral membrane is acquired in the ER (Welsch et al., 2009). Thus, the properties of the envelope may depend on the host from which the virions are released.

After internalization and uncoating (Samsa et al., 2009), the viral RNA is translated into a polyprotein which is cleaved into structural and non-structural proteins. The viral proteins synthesized such as the NS4A protein induces the ER-membrane remodeling to form membrane curvatures (Roosendaal et al., 2006; Miller et al., 2007). Consequently, the extensive membrane curvature could reduce the effective surface area and functionality of the ER (Heaton et al., 2010). Moreover, these invaginations of the ER leads to the redistribution of the FASN (Heaton et al., 2010) and the cholesterol-synthesizing enzyme, the HMGCR (Soto-Acosta et al., 2017) required for the synthesis of mevalonate, a precursor of the cholesterol, which is upregulated in response to cholesterol depletion (Goldstein and Brown, 1990). It is suggested that this mechanism occurs when NS3 recruit to FASN in the ER where the N-terminal 180 amino acids of NS3 have interaction with FASN and this association increase the FASN activity and the fatty acid biosynthesis to augment the lipid biogenesis to form the replicative complexes (Heaton et al., 2010) (Figure 1C). Moreover, the HMGCR is redistributed from the outer membrane of the nuclear envelope, where it is synthesized and localized (Pathak et al., 1986) to sites of viral replication (Peña and Harris, 2012; Soto-Acosta et al., 2017). Interestingly, although the induction of HMGCR expression during DENV infection does not occur, an increase in the activity of the enzyme due to a reduction of its phosphorylation levels was observed (Soto-Acosta et al., 2013). However, the action of viral proteins NS3 and NS4A on the HMGCR activity is unknown and the mechanism by which NS3 stimulates FASN-specific activity needs to be investigated further (Heaton et al., 2010). In summary, the presence and the increased activity of the FASN and HMGCR enzymes in the ER and their association with viral proteins such as NS3 and NS4A increase the de novo synthesis of fatty acids and cholesterol (Heaton et al., 2010; Soto-Acosta et al., 2017) (Figure 1C). This event induces the formation of the RC that comprise membrane packets (Vp), double-membrane vesicles (Ve), tubular structures (T), and convoluted membranes (CM) (Welsch et al., 2009; Junjhon et al., 2014). Inside of the Ve where the NS1, NS3, and NS5 proteins and dsRNA are localized, the RNA replication occurs (Welsch et al., 2009; Junjhon et al., 2014).

The complex set of membranes required for the viral replication is host-dependent (Perera et al., 2012). Consequently, the structures of convoluted membranes (CM) are found in DENV-infected mammalian cells (Welsch et al., 2009), but they are not induced in DENV-infected C6/36 cells (Junjhon et al., 2014; Reyes-Ruiz et al., 2018). Since the cholesterol contributes to the stability of subcellular structures, it is possible that the absence of CM could be related with the fact that the mosquitoes are cholesterol auxotrophs (CLAYTON, 1964; Rawson, 2003) and the amount of this molecule is lower in mosquito cells compared to mammalian cells. Under experimental conditions, the bovine serum from the culture medium provides the cholesterol that mosquito cells required (CLAYTON, 1964; Krebs and Lan, 2003). Considering that ~1% of unesterified cholesterol is found in the ER (Lange et al., 1999; Liscum and Munn, 1999), the cholesterol requirement during viral replication is high. Thus, viruses directly manipulate the host cell pathways involved in the uptake and biosynthesis of cholesterol to increase the levels. When the cholesterol is low in the ER, the cholesterol sensor SREBP (Sterol regulatory element binding protein)-SCAP (SREP cleavage activating protein) complex is transported to the Golgi apparatus where the cytoplasmic domain of SREBP is cleaved and the protein is translocated into the nucleus acting as a transcription factor to induce HMGCR and LDLr gene transcription (Vallett et al., 1996). It has been reported that upregulation of enzymes involved in the intermediate steps of cholesterol biosynthesis such as HMGCR and mevalonate diphospho decarboxylase (MVD) lead to higher cholesterol levels in the ER which favor the replication of WNV (Mackenzie et al., 2007) and DENV (Rothwell et al., 2009; Soto-Acosta et al., 2013, 2017). On the other hand, the modulation of exogenous cholesterol uptake by critical proteins such as the LDLr plays an essential role in flavivirus replication (Poh et al., 2012; Soto-Acosta et al., 2013). The use of drugs that inhibit cholesterol biosynthesis pathway can alter the RC formation inhibiting virus infection (Mackenzie et al., 2007; Rothwell et al., 2009; Martínez-Gutierrez et al., 2014; Soto-Acosta et al., 2017); demonstrating the essential role of the de novo synthesis of cholesterol during viral replication.

In mosquito cells little is known about the role of cholesterol during replication. However, Perera et al. demonstrated that DENV induces changes in the lipids profile of infected mosquito cells (Perera et al., 2012). Unfortunately, their mass spectrometry analysis was unable to successfully separated cholesterol. On the other hand, in Wolbachia-infected mosquito cells, it has been reported the upregulation of the apolipoprotein D, the ATP-binding cassette protein A1 (ABCA1), and a homolog of cholesterol transporter NPC2, and the downregulation of the LDLr (Geoghegan et al., 2017). All these proteins are involved in the cholesterol homeostasis. These results are exciting because the mosquitoes are cholesterol auxotrophs (Rawson, 2003) as well as the Wolbachia bacteria (Wu et al., 2004). Thus, both depend on and compete for cholesterol (Caragata et al., 2013). This competence for cholesterol is one of the reason that justify the inhibition of viral replication by Wolbachia (Moreira et al., 2009; Bian et al., 2010; van den Hurk et al., 2012; Caragata et al., 2016; Dutra et al., 2016; Geoghegan et al., 2017). However, further studies have to be performed to completely elucidate the role of cholesterol in flaviviral replication in mosquito infected cells.

The flavivirus-induced membrane rearrangements also serve as a scaffold for the assembly of the viral progeny. After uncoating, translation and genome replication, the assembly of the viral particles is carried out in the replication complexes induce in the ER. These assembly sites have a high activity of enzymes such as the cholesterol-synthesizing enzyme, HMGCR and MVD involved in the cholesterol biosynthesis pathway (Mackenzie et al., 2007; Rothwell et al., 2009; Soto-Acosta et al., 2013, 2017), which leads to the replication complexes being rich in cholesterol. The sites for viral assembly and budding have been reported as the membrane packets (Vp), which contain anchored the viral E and prM proteins to their membrane (Junjhon et al., 2014). Although the mosquito lacks the biosynthetic pathways to produce cholesterol, the Vp are present in both, mammalian and mosquito cells (Welsch et al., 2009; Junjhon et al., 2014; Reyes-Ruiz et al., 2018). The first step in the flavivirus assembly is the packaging of one copy of the viral RNA in multiple copies of the capsid (C) protein to form the nucleocapsid (Lindenbach and Rice, 2003). The viral C protein is located in a close proximity of the ER surrounding the structures called lipid droplets (LDs) (Samsa et al., 2009; Carvalho et al., 2012), which are structurally similar to circulating lipoproteins with a core of esterified lipids, where the cholesterol is an essential component (Ducharme and Bickel, 2008). The viral RNA recruitment process by the C protein to form the nucleocapsid is still unclear. However, it has been suggested that the C protein or the C protein-RNA complex is accumulated on the LDs during infection to be then mobilized to viral assembly sites (Samsa et al., 2009; Carvalho et al., 2012) (Figure 1D). Interestingly, the accumulation of C protein on LD occurs in both infected mammalian and mosquito cells, which suggest that the LD are organelles with a conserved function in the viral replication in different hosts (Samsa et al., 2009) and crucial for viral assembly. Once that the nucleocapsid is formed, this is enveloped by a lipid membrane (Kuhn et al., 2002), from the ER where are found the prM-E heterodimers to complete the assembly of immature virion and then the viral particle is transported to the Golgi complex for its maturation (Mukhopadhyay et al., 2005; Yu et al., 2008; Welsch et al., 2009).

The last step of the flaviviruses replicative cycle is the release of viral particles by exocytosis (Barrows et al., 2018). The viral particles to become infectious have to maturate by the cleavage of prM protein by the host protease furin (Pierson and Diamond, 2012) (Figure 1E). In this step, the participation of cholesterol has not been described.

On the other hand, during flavivirus infection, the non-structural protein 1 (NS1) is secreted (Scaturro et al., 2015). The NS1 protein is one of the most enigmatic proteins of flaviviruses because it is the only protein that is secreted, playing essential roles in immune evasion, pathogenesis and viral replication (Scaturro et al., 2015). The reported crystal structure of the secreted DENV NS1 protein revealed its peculiar three-dimensional fold as hexamer that forms a lipoprotein particle with an open-barrel protein shell and a central channel rich in lipids, reminiscent of the composition of high-density lipoprotein (Gutsche et al., 2011). NS1-associated lipid species include triglycerides, as well as cholesterol and phospholipid esters, a composition that evokes plasma lipoproteins in humans involved in vascular homeostasis. Thus, the discovery that DENV NS1 carries lipids could have critical pathophysiological implications during the disease (Gutsche et al., 2011) and supports the idea that cholesterol is required for the secretion of NS1 (Figure 1E).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.