**2. A lipid perspective of the virus life cycle**

Inherent to their condition of obligate intracellular parasites, viruses have to invade a cell to complete their replicative cycle. During this step, viruses express their own proteins and also co-opt host cell factors for multiplication, including lipids [15]. A schematic view of a virus replication cycle is shown in Figure 1. Initial steps of viral infection include the attachment of the virus particle to a specific receptor located on the cell surface, in some

© 2013 Sobrino et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cases a specific lipid (section 2.1.1). The viral genome has to entry into the host cell to reach the replication sites. Different lipids, located either on plasma and/or endosomal membranes, can contribute to these processes by enabling receptor clustering, virus internalization, or membrane fusion (sections 2.1.2 and 2.1.3). Replication of viral genome can take place associated to cellular membranes or other lipid structures, like lipid droplets, forming structures termed replication complexes (section 2.2). Newly synthesized viral genomes are enclosed inside *de novo* synthesized viral particles, a process in which several lipids can play, again, an important role (section 2.3), especially in the case of viruses containing a lipid envelope as an integral component of their infectious particle. Then, viral particles maturate to render infectious particles that are released from host cell to initiate a new infection cycle.

Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 293

**Virus Lipid Function Refs.**  VSV phosphatidylserine Cellular receptor [17] LBPA Cofactor for membrane fusion [18,19] SV40 GM1 Cellular receptor [20] cholesterol Lipid raft-caveola mediated endocytosis [21] DENV LBPA Cofactor for membrane fusion [22] VACV phosphatidylserine Induction of viral internalization [23] Abbreviations used in this Table: DENV, dengue virus; LBPA, lysobisphosphatidic acid; SV40, simian virus 40; VACV,

The first event of virus infection comprises the recognition of the target cell, which generally occurs through the interaction between the virus and a specific receptor on the cell surface. Receptors exploited by viruses include different macromolecules like proteins, carbohydrates and lipids. An increasing number of viruses is known to attach to lipidcontaining molecules. For instance, members of the *Polyomaviridae* family use gangliosides, being the binding highly specific [24]. The simian virus 40 (SV40) employs exclusively the ganglioside GM1, whereas the mouse polyomavirus can use GD1a and GT1b, and BK virus can utilize GD1b and GT1b [25-28]. Other important human pathogens, such as influenza virus (*Orthomyxoviridae*) and Human immunodeficiency virus, HIV (*Retroviridae*), can also

Another example of a virus whose receptor is supposed to be a lipid is the rhabdovirus vesicular stomatitis virus (VSV), which seems to gain cell entry through interaction with negatively charged phospholipids, like phosphatidylserine [17]. VSV particles interact very strongly with membranes containing phosphatidylserine through viral glycoprotein G [31], and although it is not actually clear whether phosphatidylserine is the viral receptor [32], a direct interaction between the G protein and this lipid could take place in the membrane

On the other hand, some members of the *Flaviviridae* family -hepatitis C virus (HCV), GB virus C/hepatitis G virus and bovine viral diarrhea virus (BVDV)-, use the low-density lipoprotein receptor (LDL-R) [33], which is a cholesterol receptor. An interesting case is hepatitis C virus (HCV) that requires the interaction with the low-density lipoprotein receptor (LDL-R) and with glycosaminoglycans to entry into the cell [8]. The component of the virion that interacts with LDL-R likely is a cell-derived lipoprotein, i. e. a viral-

Cells use a broad spectrum of mechanisms to internalize substances from their environment. Endocytosis is a general term for the internalization of particles, solutes, fluids, and membrane components by invagination of the plasma membrane and internalization of the

vaccinia virus; VSV, vesicular stomatitis virus.

*2.1.1. Lipids and viral attachment* 

bind to different gangliosides [29,30].

[31].

lipoprotein (section 2.3.1).

*2.1.2. Lipids and viral internalization* 

**Table 1.** Examples of lipids required for viral entry

**Figure 1.** Schematic view of a virus replication cycle. For a detailed description of the different roles of lipids during virus infection see the text.

#### **2.1. Lipids and viral entry**

The viral entry into a host cell to start their replicative cycle involves the attachment of the virus particle to a specific receptor(s) located on the cell surface, prior to the introduction of the viral genome within the host cell. The latter can take place by internalization of the whole viral particle, constituting a sort of minute Trojan horse [16], or by direct penetration of viral genome from plasma membrane. During these processes, a variety of specific lipids play multiple roles, which may vary between viruses (Table 1).


Abbreviations used in this Table: DENV, dengue virus; LBPA, lysobisphosphatidic acid; SV40, simian virus 40; VACV, vaccinia virus; VSV, vesicular stomatitis virus.

**Table 1.** Examples of lipids required for viral entry

#### *2.1.1. Lipids and viral attachment*

292 Lipid Metabolism

to initiate a new infection cycle.

Host cell

Viral genome release

Virus entry

Attachment

Virus

Expression of viral factors

lipids during virus infection see the text.

Cytoplasm

**2.1. Lipids and viral entry** 

cases a specific lipid (section 2.1.1). The viral genome has to entry into the host cell to reach the replication sites. Different lipids, located either on plasma and/or endosomal membranes, can contribute to these processes by enabling receptor clustering, virus internalization, or membrane fusion (sections 2.1.2 and 2.1.3). Replication of viral genome can take place associated to cellular membranes or other lipid structures, like lipid droplets, forming structures termed replication complexes (section 2.2). Newly synthesized viral genomes are enclosed inside *de novo* synthesized viral particles, a process in which several lipids can play, again, an important role (section 2.3), especially in the case of viruses containing a lipid envelope as an integral component of their infectious particle. Then, viral particles maturate to render infectious particles that are released from host cell

**Figure 1.** Schematic view of a virus replication cycle. For a detailed description of the different roles of

Viral replication

Mitochondrion

Virus-induced replication complex

Nucleus

Virus assembly and release

> Golgi complex

Endoplasmic reticulum

The viral entry into a host cell to start their replicative cycle involves the attachment of the virus particle to a specific receptor(s) located on the cell surface, prior to the introduction of the viral genome within the host cell. The latter can take place by internalization of the whole viral particle, constituting a sort of minute Trojan horse [16], or by direct penetration of viral genome from plasma membrane. During these processes, a variety of specific lipids

play multiple roles, which may vary between viruses (Table 1).

The first event of virus infection comprises the recognition of the target cell, which generally occurs through the interaction between the virus and a specific receptor on the cell surface. Receptors exploited by viruses include different macromolecules like proteins, carbohydrates and lipids. An increasing number of viruses is known to attach to lipidcontaining molecules. For instance, members of the *Polyomaviridae* family use gangliosides, being the binding highly specific [24]. The simian virus 40 (SV40) employs exclusively the ganglioside GM1, whereas the mouse polyomavirus can use GD1a and GT1b, and BK virus can utilize GD1b and GT1b [25-28]. Other important human pathogens, such as influenza virus (*Orthomyxoviridae*) and Human immunodeficiency virus, HIV (*Retroviridae*), can also bind to different gangliosides [29,30].

Another example of a virus whose receptor is supposed to be a lipid is the rhabdovirus vesicular stomatitis virus (VSV), which seems to gain cell entry through interaction with negatively charged phospholipids, like phosphatidylserine [17]. VSV particles interact very strongly with membranes containing phosphatidylserine through viral glycoprotein G [31], and although it is not actually clear whether phosphatidylserine is the viral receptor [32], a direct interaction between the G protein and this lipid could take place in the membrane [31].

On the other hand, some members of the *Flaviviridae* family -hepatitis C virus (HCV), GB virus C/hepatitis G virus and bovine viral diarrhea virus (BVDV)-, use the low-density lipoprotein receptor (LDL-R) [33], which is a cholesterol receptor. An interesting case is hepatitis C virus (HCV) that requires the interaction with the low-density lipoprotein receptor (LDL-R) and with glycosaminoglycans to entry into the cell [8]. The component of the virion that interacts with LDL-R likely is a cell-derived lipoprotein, i. e. a virallipoprotein (section 2.3.1).

#### *2.1.2. Lipids and viral internalization*

Cells use a broad spectrum of mechanisms to internalize substances from their environment. Endocytosis is a general term for the internalization of particles, solutes, fluids, and membrane components by invagination of the plasma membrane and internalization of the resulting membrane vesicles [24,34-36]. The plasma membrane does not present a continuous or homogeneous composition. It contains lipid microdomains termed lipid rafts [37], characterized by their high content of cholesterol, glycosphinglolipids, glycophosphatidylinositol (GPI), anchored proteins like the GPI-anchored, myristoylated and palmytoylated proteins, as well as transmembrane proteins [38]. Lipid rafts have been associated with various endocytic mechanisms to internalize these membrane regions [39- 42], being the formation of cave-shaped invaginations, termed caveolae, the predominant mechanism [24,34]. Lipid rafts have been related to the entry of a number of viruses, for example the coronavirus severe acute respiratory syndrome (SARS), murine leukemia virus, herpes simplex virus, Japanese encephalitis virus, SV40, and echovirus 1 [34,43-48]. In addition, some viruses require cholesterol-enriched microdomains in the viral membrane for efficient virus entry, for example influenza virus A, human herpes virus 6, and Canine distemper virus [49-51]. On the other hand, some viruses that enter into the cells using mechanisms independent of lipid rafts require cholesterol for an efficient internalization. This is the case of foot-and-mouth disease virus (FMDV) and human rhinovirus type 2, whose entry into cells, by clathrin-mediated endocytosis, requires the presence of plasma membrane cholesterol [52,53]. In other viruses such as HIV-1, a requirement of cholesterol for viral entry has been documented [47] and related to the clustering of viral receptors, thus enabling viral internalization [54]. This role of cholesterol and lipid-rafts has also been documented for coxsackievirus B3 (CVB3) infection [55].

Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 295

[63-66]. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or, in the case of viruses entering through receptor-mediated endocytosis, with membranes of endocytic organelles in which particles are internalized. There is evidence showing that both groups of viruses use fusion proteins that, via hydrophobic segments, interact with membrane lipids, leading to conformational changes that make them able for fusion [63-66]. Compelling evidence indicates that specific lipids can influence the compartment of virus uncoating and viral genome delivery into the cytosol [22,67,68]. A number of enveloped viruses take advantage of the low pH inside endosomes to promote endosome fusion, permitting viral genome release [69]. Thus, utilization of specific lipids allows the virus to ensure membrane fusion at the proper cellular compartment. For instance, DENV takes advantage of the anionic late endosome-specific lipid bis(monoacylglycero)phosphate (BMP), also named lysobisphosphatidic acid (LBPA), to promote virus fusion with the late endosomal membrane [22]. A relevant role of LBPA in promoting membrane fusion and lipid mixing has also been shown in VSV infection [70]. Initially, VSV envelope fuses with intraluminal vesicles inside multivesicular bodies, which later fuse with external membrane of the multivesicular body, allowing the release of viral nucleocapsid to the cytosol [18,19]. However, fusion of other viruses, such as influenza virus, does not rely on these lipids [70]. Cholesterol, a major and vital constituent of eukaryotic cellular membranes, has been implicated in promoting lipid transfer and fusion pore expansion in the virus-cell membrane fusion mediated by the haemagglutinin of influenza virus [68]. The presence of cholesterol on the target membrane also promotes West Nile virus (WNV) membrane fusion activity [71], and both cholesterol and sphingolipids, but not lipid-rafts, are required for

Regarding the entry of non-enveloped viruses, it is generally believed that the mechanism(s) involved does not include membrane fusion activity. Nevertheless, recent data obtained from biochemical and structural studies indicate that the overall mechanisms of entry of certain non-enveloped viruses are similar to those of enveloped ones, and that capsid proteins can function in these activities in a manner similar to that of the membrane viral proteins [72]. For instance, the outer capsid protein VP5 of the non-enveloped rotaviruses and orbiviruses, shares secondary structural features with fusion proteins of enveloped viruses [73], like the capacity to associate with lipid rafts in cellular membranes [72]. These findings indicate that VP5 may be responsible for membrane penetration [74]. Post-translational modifications of viral proteins, i.e. myristoylation of capsid protein VP4 in poliovirus (PV) and VP2 of polyomavirus, have been related to the ability of these proteins to induce pores on cellular membranes for

Following entry into the host cell, viruses have to produce accurate self-copies to generate new infectious viral particles. To this end, viruses use to recruit cellular factors, including

alphavirus fusion [67].

genome release [75,76].

**2.2. Lipids and viral multiplication** 

lipids and enzymes involved in their metabolism.

The plasma membrane also exhibits clusters of other lipids like phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) [56], which is a minor lipid of the inner leaflet of the plasma membrane with an important role in the clathrin-mediated endocytosis [57-60]. Even when the number of viruses that use clathrin-mediated endocytosis to entry into the cells is wide [61], the importance of this lipid in viral entry has not been analyzed in depth yet. However, it has been reported that PI(4,5)P2 production by a specific lipid kinase is crucial for HIV-1 entry in permissive lymphocytes [62]. Likewise, foot-and-mouth disease virus (FMDV) and VSV require the presence of this phospholipid in the plasma membrane for internalization (Vázquez-Calvo *et al*., submitted).

As commented before, specific lipids located in the viral particles can also play a role on viral entry of enveloped viruses [8], including 'those located in' lipid rafts [49-51]. Vaccinia virus provides another example of the relationships between lipids located on the viral particle and viral entry. In this case, the presence of exposed phosphatidylserine in the viral envelope is critical to induce blebs on cellular membrane that promote virus internalization [23].

#### *2.1.3. Lipids and viral genome delivery*

Viruses have to release their genome from the particle to enable proper expression of viral proteins and genome replication within host cell. In the case of enveloped viruses, fusion between viral envelope and cellular membranes is a generalized strategy to facilitate these events. This process is assisted by viral proteins termed fusion proteins, and results in lipid mixing between the viral envelope and the target cellular membrane [63-66]. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or, in the case of viruses entering through receptor-mediated endocytosis, with membranes of endocytic organelles in which particles are internalized. There is evidence showing that both groups of viruses use fusion proteins that, via hydrophobic segments, interact with membrane lipids, leading to conformational changes that make them able for fusion [63-66]. Compelling evidence indicates that specific lipids can influence the compartment of virus uncoating and viral genome delivery into the cytosol [22,67,68]. A number of enveloped viruses take advantage of the low pH inside endosomes to promote endosome fusion, permitting viral genome release [69]. Thus, utilization of specific lipids allows the virus to ensure membrane fusion at the proper cellular compartment. For instance, DENV takes advantage of the anionic late endosome-specific lipid bis(monoacylglycero)phosphate (BMP), also named lysobisphosphatidic acid (LBPA), to promote virus fusion with the late endosomal membrane [22]. A relevant role of LBPA in promoting membrane fusion and lipid mixing has also been shown in VSV infection [70]. Initially, VSV envelope fuses with intraluminal vesicles inside multivesicular bodies, which later fuse with external membrane of the multivesicular body, allowing the release of viral nucleocapsid to the cytosol [18,19]. However, fusion of other viruses, such as influenza virus, does not rely on these lipids [70]. Cholesterol, a major and vital constituent of eukaryotic cellular membranes, has been implicated in promoting lipid transfer and fusion pore expansion in the virus-cell membrane fusion mediated by the haemagglutinin of influenza virus [68]. The presence of cholesterol on the target membrane also promotes West Nile virus (WNV) membrane fusion activity [71], and both cholesterol and sphingolipids, but not lipid-rafts, are required for alphavirus fusion [67].

Regarding the entry of non-enveloped viruses, it is generally believed that the mechanism(s) involved does not include membrane fusion activity. Nevertheless, recent data obtained from biochemical and structural studies indicate that the overall mechanisms of entry of certain non-enveloped viruses are similar to those of enveloped ones, and that capsid proteins can function in these activities in a manner similar to that of the membrane viral proteins [72]. For instance, the outer capsid protein VP5 of the non-enveloped rotaviruses and orbiviruses, shares secondary structural features with fusion proteins of enveloped viruses [73], like the capacity to associate with lipid rafts in cellular membranes [72]. These findings indicate that VP5 may be responsible for membrane penetration [74]. Post-translational modifications of viral proteins, i.e. myristoylation of capsid protein VP4 in poliovirus (PV) and VP2 of polyomavirus, have been related to the ability of these proteins to induce pores on cellular membranes for genome release [75,76].

#### **2.2. Lipids and viral multiplication**

294 Lipid Metabolism

resulting membrane vesicles [24,34-36]. The plasma membrane does not present a continuous or homogeneous composition. It contains lipid microdomains termed lipid rafts [37], characterized by their high content of cholesterol, glycosphinglolipids, glycophosphatidylinositol (GPI), anchored proteins like the GPI-anchored, myristoylated and palmytoylated proteins, as well as transmembrane proteins [38]. Lipid rafts have been associated with various endocytic mechanisms to internalize these membrane regions [39- 42], being the formation of cave-shaped invaginations, termed caveolae, the predominant mechanism [24,34]. Lipid rafts have been related to the entry of a number of viruses, for example the coronavirus severe acute respiratory syndrome (SARS), murine leukemia virus, herpes simplex virus, Japanese encephalitis virus, SV40, and echovirus 1 [34,43-48]. In addition, some viruses require cholesterol-enriched microdomains in the viral membrane for efficient virus entry, for example influenza virus A, human herpes virus 6, and Canine distemper virus [49-51]. On the other hand, some viruses that enter into the cells using mechanisms independent of lipid rafts require cholesterol for an efficient internalization. This is the case of foot-and-mouth disease virus (FMDV) and human rhinovirus type 2, whose entry into cells, by clathrin-mediated endocytosis, requires the presence of plasma membrane cholesterol [52,53]. In other viruses such as HIV-1, a requirement of cholesterol for viral entry has been documented [47] and related to the clustering of viral receptors, thus enabling viral internalization [54]. This role of cholesterol and lipid-rafts has also been

The plasma membrane also exhibits clusters of other lipids like phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) [56], which is a minor lipid of the inner leaflet of the plasma membrane with an important role in the clathrin-mediated endocytosis [57-60]. Even when the number of viruses that use clathrin-mediated endocytosis to entry into the cells is wide [61], the importance of this lipid in viral entry has not been analyzed in depth yet. However, it has been reported that PI(4,5)P2 production by a specific lipid kinase is crucial for HIV-1 entry in permissive lymphocytes [62]. Likewise, foot-and-mouth disease virus (FMDV) and VSV require the presence of this phospholipid in the plasma membrane for internalization

As commented before, specific lipids located in the viral particles can also play a role on viral entry of enveloped viruses [8], including 'those located in' lipid rafts [49-51]. Vaccinia virus provides another example of the relationships between lipids located on the viral particle and viral entry. In this case, the presence of exposed phosphatidylserine in the viral envelope is

Viruses have to release their genome from the particle to enable proper expression of viral proteins and genome replication within host cell. In the case of enveloped viruses, fusion between viral envelope and cellular membranes is a generalized strategy to facilitate these events. This process is assisted by viral proteins termed fusion proteins, and results in lipid mixing between the viral envelope and the target cellular membrane

critical to induce blebs on cellular membrane that promote virus internalization [23].

documented for coxsackievirus B3 (CVB3) infection [55].

(Vázquez-Calvo *et al*., submitted).

*2.1.3. Lipids and viral genome delivery* 

Following entry into the host cell, viruses have to produce accurate self-copies to generate new infectious viral particles. To this end, viruses use to recruit cellular factors, including lipids and enzymes involved in their metabolism.

#### *2.2.1. Cellular membranes and viral replication complex assembly*

Viruses co-opt host cell factors to develop the most adequate environment for their replication, a feature that is especially highlighted by the viral replication complex found assembled inside cells infected with positive strand RNA viruses [2,15,77]. Viruses belonging to this group share as a common feature a viral genome consisting of one or more RNA molecules of positive polarity that mimic the characteristics of cellular messenger RNA (mRNA) to be translated into viral proteins. Positive strand RNA viruses comprise several viral families that include important animal (including human) and plant pathogens such as *Picornaviridae* (i.e. PV, FMDV), *Flaviviridae* (i.e. DENV, WNV, HCV), *Caliciviridae* (i.e. Nowalk virus), *Coronaviridae* (i.e. SARS coronavirus), or *Togaviridae* (i.e. rubella virus). Replication of positive-strand RNA viruses is tightly associated to intracellular lipid membranes derived from different organelles: endoplasmic reticulum, Golgi complex, mitochondria, chloroplasts, peroxisomes, vacuoles, endosomes, or lysosomes [2]. Besides membranes derived from cellular organelles, these viruses can also usurp cytoplasmic lipid droplets for their replication [78,79]. In this way, viral replication results in the induction of marked alterations of the intracellular architecture mainly characterized by the remodelling of cellular membranes. These alterations include intracellular membrane proliferation and changes on shape and size of membranous structures. Consequently, viral replication originates a variety of structures that may rely on different mechanisms for their generation [12,15]. Examples of these structures (Figure 2) include the formation of convoluted membranes and vesicle packets as a result of flavivirus replication [80-82], the development of heterogeneous vesicular structures that conform the membranous web found in HCV-infected cells [83], or the proliferation of vesicular structures (including double membrane vesicles) in cells infected by enteroviruses (a genus within the *Picornaviridae* family) like PV [84,85].

Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 297

**Figure 2.** Distinct alterations on intracellular membrane architecture induced by the infection of positive strand RNA viruses. A) Induction of convoluted membranes and vesicle packets in flavivirus infected cells. Image corresponds to Vero cells infected with WNV, fixed and processed for transmission electron microscopy at 24 h post-infection. B) Proliferation of vesicular structures in enterovirusinfected cells. Porcine cells (IBRS-2) were infected with the enterovirus swine vesicular disease virus (SVDV), fixed, and processed for transmission electron microscopy at 7 h post-infection. For technical

dependence on different cellular lipids for replication complex organization, although their

To render the specific lipid microenvironment adequate for multiplication, viruses co-opt cellular machinery for their replication, including host factors involved in different aspects of lipid metabolism, i.e. sterol biosynthesis, fatty acid metabolism and synthesis of specific phosphoinositides [15]. For instance, a marked alteration of cellular metabolism and an increase in fatty acid biosynthetic pathway have been described upon human cytomegalovirus (HCMV), DENV or HCV infection [1,5,6,11]. The association of viral multiplication with modulation of host cell factors involved in lipid metabolism is not an exclusive feature of animal viruses, thus, replication and recombination of the plant pathogens tombusviruses has been revealed to rely on host genes involved in lipid metabolism [96-99]. Representative examples of cellular factors related to lipid metabolism

Several studies have highlighted the role of the cholesterol and the cholesterol biosynthetic pathway in the replication of viruses, including important human pathogens belonging to the *Flaviviridae* family -WNV [90], DENV [110], and HCV [8,111-113]- and to the *Caliciviridae* -Nowalk virus [114]-families. In addition, the cholesterol biosynthetic pathway has also been

details related to virus infection and sample preparation see references [81,84].

*2.2.2. Cellular lipids involved in viral replication complex assembly* 

and associated to viral replication are summarized in Table 2.

roles and importance vary between viruses.

Morphological changes on membrane shape induced by viral infections are accompanied by an enrichment in the viral and cellular components, including specific proteins and lipids [1- 3,12]. Despite the diversity of the membrane alterations induced, these changes provide the physical scaffold for viral replication, thus offering the most suitable platform for viral replication complex assembly, and hence increasing the local concentration of specific cellular and viral factors necessary for replication [1,12]. In addition, membrane remodelling can also improve viral multiplication by hiding viral components from the innate immune system [1,12,86]. In flaviviruses (DENV and WNV) the evasion of interferon response has been shown to depend on the expression of hydrophobic viral proteins involved in membrane rearrangements [87-89]; in particular, the cholesterol content of these membranes is important to down regulate the interferon-stimulated antiviral signalling response to infection [90]. Related to this, antiviral interferon response also involves down regulation of sterol biosynthesis [91]. Likewise, lipid droplets, which can constitute platforms for viral replication, also play important roles on the coordination of immune responses [92].

All these changes in the membrane morphology and composition result in the formation of customized cellular microenvironments that support viral replication and can be actually considered novel virus-induced organelles [93-95]. Regarding the lipid composition of these structures, great progresses have been recently made (see below) that have uncovered the

296 Lipid Metabolism

*2.2.1. Cellular membranes and viral replication complex assembly* 

Viruses co-opt host cell factors to develop the most adequate environment for their replication, a feature that is especially highlighted by the viral replication complex found assembled inside cells infected with positive strand RNA viruses [2,15,77]. Viruses belonging to this group share as a common feature a viral genome consisting of one or more RNA molecules of positive polarity that mimic the characteristics of cellular messenger RNA (mRNA) to be translated into viral proteins. Positive strand RNA viruses comprise several viral families that include important animal (including human) and plant pathogens such as *Picornaviridae* (i.e. PV, FMDV), *Flaviviridae* (i.e. DENV, WNV, HCV), *Caliciviridae* (i.e. Nowalk virus), *Coronaviridae* (i.e. SARS coronavirus), or *Togaviridae* (i.e. rubella virus). Replication of positive-strand RNA viruses is tightly associated to intracellular lipid membranes derived from different organelles: endoplasmic reticulum, Golgi complex, mitochondria, chloroplasts, peroxisomes, vacuoles, endosomes, or lysosomes [2]. Besides membranes derived from cellular organelles, these viruses can also usurp cytoplasmic lipid droplets for their replication [78,79]. In this way, viral replication results in the induction of marked alterations of the intracellular architecture mainly characterized by the remodelling of cellular membranes. These alterations include intracellular membrane proliferation and changes on shape and size of membranous structures. Consequently, viral replication originates a variety of structures that may rely on different mechanisms for their generation [12,15]. Examples of these structures (Figure 2) include the formation of convoluted membranes and vesicle packets as a result of flavivirus replication [80-82], the development of heterogeneous vesicular structures that conform the membranous web found in HCV-infected cells [83], or the proliferation of vesicular structures (including double membrane vesicles) in

cells infected by enteroviruses (a genus within the *Picornaviridae* family) like PV [84,85].

replication, also play important roles on the coordination of immune responses [92].

All these changes in the membrane morphology and composition result in the formation of customized cellular microenvironments that support viral replication and can be actually considered novel virus-induced organelles [93-95]. Regarding the lipid composition of these structures, great progresses have been recently made (see below) that have uncovered the

Morphological changes on membrane shape induced by viral infections are accompanied by an enrichment in the viral and cellular components, including specific proteins and lipids [1- 3,12]. Despite the diversity of the membrane alterations induced, these changes provide the physical scaffold for viral replication, thus offering the most suitable platform for viral replication complex assembly, and hence increasing the local concentration of specific cellular and viral factors necessary for replication [1,12]. In addition, membrane remodelling can also improve viral multiplication by hiding viral components from the innate immune system [1,12,86]. In flaviviruses (DENV and WNV) the evasion of interferon response has been shown to depend on the expression of hydrophobic viral proteins involved in membrane rearrangements [87-89]; in particular, the cholesterol content of these membranes is important to down regulate the interferon-stimulated antiviral signalling response to infection [90]. Related to this, antiviral interferon response also involves down regulation of sterol biosynthesis [91]. Likewise, lipid droplets, which can constitute platforms for viral **Figure 2.** Distinct alterations on intracellular membrane architecture induced by the infection of positive strand RNA viruses. A) Induction of convoluted membranes and vesicle packets in flavivirus infected cells. Image corresponds to Vero cells infected with WNV, fixed and processed for transmission electron microscopy at 24 h post-infection. B) Proliferation of vesicular structures in enterovirusinfected cells. Porcine cells (IBRS-2) were infected with the enterovirus swine vesicular disease virus (SVDV), fixed, and processed for transmission electron microscopy at 7 h post-infection. For technical details related to virus infection and sample preparation see references [81,84].

dependence on different cellular lipids for replication complex organization, although their roles and importance vary between viruses.

#### *2.2.2. Cellular lipids involved in viral replication complex assembly*

To render the specific lipid microenvironment adequate for multiplication, viruses co-opt cellular machinery for their replication, including host factors involved in different aspects of lipid metabolism, i.e. sterol biosynthesis, fatty acid metabolism and synthesis of specific phosphoinositides [15]. For instance, a marked alteration of cellular metabolism and an increase in fatty acid biosynthetic pathway have been described upon human cytomegalovirus (HCMV), DENV or HCV infection [1,5,6,11]. The association of viral multiplication with modulation of host cell factors involved in lipid metabolism is not an exclusive feature of animal viruses, thus, replication and recombination of the plant pathogens tombusviruses has been revealed to rely on host genes involved in lipid metabolism [96-99]. Representative examples of cellular factors related to lipid metabolism and associated to viral replication are summarized in Table 2.

Several studies have highlighted the role of the cholesterol and the cholesterol biosynthetic pathway in the replication of viruses, including important human pathogens belonging to the *Flaviviridae* family -WNV [90], DENV [110], and HCV [8,111-113]- and to the *Caliciviridae* -Nowalk virus [114]-families. In addition, the cholesterol biosynthetic pathway has also been

#### 298 Lipid Metabolism


Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 299

its replication is based on the outer mitochondrial membrane and is dependent on the anionic phospholipid cardiolipin, which is almost exclusive of these membranes [120]. In this regard, more than 20 years ago, phospholipids were already associated to the replication of PV, a member of the *Picornaviridae* family [121]. More recently, this relationship has been confirmed after the identification of a specific phospholipid, the phosphatidylinositol 4 phosphate (PI4P), as a key component of PV replication complexes [93]. Requirement of PI4P is shared by other members of the *Picornaviridae* family - CVB3, Aichi virus (AiV), bovine kubovirus, and human rhinovirus 14 [93,108,109,122]- and also by viruses from other families, i.e. HCV [93,106,107,123-126]. All these viruses can specifically recruit different isoforms of the enzyme that drives the formation of PI4P from phosphatidylinositol, the phosphatidylinositol 4-kinase class III (PI4KIII) α or β, to their replication complexes. For instance, HCV recruits the lipid kinase PI4KIIIα by direct interaction with viral protein NS5A [125,127], while in picornaviruses, the recruitment of PI4KIIIβ can be mediated by the interaction of viral protein 3A with a third cellular partner associated to the viral replication complex, ABC3D (acyl-coenzyme A binding domain containing 3) [108] or other proteins implicated in the secretory pathway [93]. The dependence on either PI4KIIIα or β isoforms varies between viruses. Replication of picornaviruses is specifically associated to PI4P synthesized by PI4KIIIβ [93,108,109,122], while replication of HCV has been mainly associated to the function of PI4KIIIα [106,107], and in a lower extent to PI4KIIIβ [93,126]. In any case, PI4P is not universally required among viruses, since the replication of the flaviviruses (WNV and DENV), and the pestivirus bovine viral diarrhea virus (all members of the *Flaviviridae* family, like HCV) has

The presence of specific lipids in the viral replication complex can accomplish with several missions. For instance, post-translational modification of viral proteins by lipids is associated to viral replication functions [128,129]. Table 3 displays representative examples

**Virus Lipid Function Refs.**  BMV fatty acids Increase in membrane plasticity and fluidity [100,101] DENV fatty acids Energy production to support viral replication [5] PV PI4P Anchor of viral replicase to replication complex [93] FHV cardiolipin Anchor of viral replicase to replication complex [120] HCV sphingomyelin Activation of RNA polymerase activity [130] WNV cholesterol Innate immune evasion [90] Abbreviations used in this table: BMV, brome mosaic virus; HCV, hepatitis C virus; DENV, Dengue virus; FHV, flock

Lipids can contribute to viral replication by acting as scaffolding molecules to anchor viral proteins. In PV, location of specific phospholipids (PI4P) to the viral replication sites

house virus; PI4P, phosphatidylinositol 4-phosphate; PV, poliovirus; WNV, West Nile virus.

been shown to be independent of PI4P [81,106,125].

of lipid functions during viral replication.

**Table 3.** Examples of lipid roles during viral replication

*2.2.3. Lipid functions associated to viral genome replication* 

Abbreviations used in this Table: AiV, Aichi virus; BMV: brome mosaic virus, CVB3, coxsackievirus B3; DCV, Droshophila C virus; DENV, Dengue virus; Erg25, ergosterol enzyme 25; FASN, fatty acid synthase; HCV, Hepatitis C virus; 3-HMG-CoA reductase, 3-hydroxy-methyglutaryl-CoA reductase; INO2, inositol-1-phosphate synthase 2; Ole1, Delta(9) fatty acid desaturase; PI4KIIIα and β, phosphatidylinositol 4-kinase class III α and β; PI4P, phosphatidylinositol 4-phosphate; PV, poliovirus; SMO1 and 2, sterol4α-methyl-oxidase 1 and 2; SREBP, sterol regulatory element binding protein; TBSV, tomato bushy stunt virus; WNV, West Nile virus.

**Table 2.** Examples of host cell genes associated to lipid metabolism and involved in viral replication

associated to the infection of animal pathogens like African swine fever virus [115]. On the other hand, sterols have been involved in the replication of plant pathogens, for example tomato bushy stunt virus (TBSV) [104]. Due to the high diversity of viruses that exploit the cholesterol biosynthetic pathway for replication, this could consider a common requirement. However, replication of viruses may rely on lipids other than cholesterol, as described for the alphanodavirus flock house virus (FHV) [116].

Another major class of lipids that has been related to viral replication are the fatty acids, whose metabolism has been shown to be required for the multiplication of viruses such as brome mosaic virus (BMV) [100,101], Droshophila C virus (DCV) [103], CVB3 [117], and PV [118]. In some cases, in addition to the dependence of cholesterol (discussed in the previous paragraph), viral multiplication is also dependent on fatty acid synthesis. Examples of viruses sharing both cholesterol and fatty acid requirements include DENV [102], WNV [81,102], and HCV [8,112]. Indeed, during DENV infection, the key enzyme responsible for fatty acid synthesis, the fatty acid synthase (FASN), is recruited to the viral replication complex by direct interaction with the viral protein NS3, enhancing its activity [102]. Dependence of DENV replication on fatty acids is shared by mammalian and mosquito host cells [9]. Even more, DENV modulates lipid metabolism through induction of a form of autophagy that targets lipid droplet stores, promoting the depletion of cellular triglycerides and the release of fatty acids. This results on an increase in β-oxidation and ATP production that stimulate viral replication [1,5]. Infection with other viruses (HCV or CVB3) also relies on fatty acids and results in an increase on FASN expression [117,119], a phenomenon that does not occur upon DENV or WNV infection [81,102].

Besides cholesterol and fatty acids, specific phospholipids can also play a key role in viral replication. For instance, replication of TBSV and FHV is dependent on phospholipid biosynthesis [105]. Replication of FHV was initially associated to glycerophospholipids, being independent of cholesterol or sphingomyelin (a membrane phospholipid that is not derived from glycerol) [116]. However, recent advances on the biology of FHV indicate that its replication is based on the outer mitochondrial membrane and is dependent on the anionic phospholipid cardiolipin, which is almost exclusive of these membranes [120]. In this regard, more than 20 years ago, phospholipids were already associated to the replication of PV, a member of the *Picornaviridae* family [121]. More recently, this relationship has been confirmed after the identification of a specific phospholipid, the phosphatidylinositol 4 phosphate (PI4P), as a key component of PV replication complexes [93]. Requirement of PI4P is shared by other members of the *Picornaviridae* family - CVB3, Aichi virus (AiV), bovine kubovirus, and human rhinovirus 14 [93,108,109,122]- and also by viruses from other families, i.e. HCV [93,106,107,123-126]. All these viruses can specifically recruit different isoforms of the enzyme that drives the formation of PI4P from phosphatidylinositol, the phosphatidylinositol 4-kinase class III (PI4KIII) α or β, to their replication complexes. For instance, HCV recruits the lipid kinase PI4KIIIα by direct interaction with viral protein NS5A [125,127], while in picornaviruses, the recruitment of PI4KIIIβ can be mediated by the interaction of viral protein 3A with a third cellular partner associated to the viral replication complex, ABC3D (acyl-coenzyme A binding domain containing 3) [108] or other proteins implicated in the secretory pathway [93]. The dependence on either PI4KIIIα or β isoforms varies between viruses. Replication of picornaviruses is specifically associated to PI4P synthesized by PI4KIIIβ [93,108,109,122], while replication of HCV has been mainly associated to the function of PI4KIIIα [106,107], and in a lower extent to PI4KIIIβ [93,126]. In any case, PI4P is not universally required among viruses, since the replication of the flaviviruses (WNV and DENV), and the pestivirus bovine viral diarrhea virus (all members of the *Flaviviridae* family, like HCV) has been shown to be independent of PI4P [81,106,125].

#### *2.2.3. Lipid functions associated to viral genome replication*

298 Lipid Metabolism

**Virus Host factor Function Refs.**  BMV Ole1 Fatty acid desaturation [100,101] DENV FASN Fatty acid synthesis [102] DCV SREBP Fatty acid synthesis [103] WNV 3-HMG-CoA reductase Cholesterol synthesis [90] TBSV Erg25, SMO1, SMO2 Sterol synthesis [104]

HCV PI4KIIIα Synthesis of PI4P [106,107] PV, CVB3, AiV PI4KIIIβ Synthesis of PI4P [93,108,109]

Abbreviations used in this Table: AiV, Aichi virus; BMV: brome mosaic virus, CVB3, coxsackievirus B3; DCV, Droshophila C virus; DENV, Dengue virus; Erg25, ergosterol enzyme 25; FASN, fatty acid synthase; HCV, Hepatitis C virus; 3-HMG-CoA reductase, 3-hydroxy-methyglutaryl-CoA reductase; INO2, inositol-1-phosphate synthase 2; Ole1,

phosphatidylinositol 4-phosphate; PV, poliovirus; SMO1 and 2, sterol4α-methyl-oxidase 1 and 2; SREBP, sterol

**Table 2.** Examples of host cell genes associated to lipid metabolism and involved in viral replication

associated to the infection of animal pathogens like African swine fever virus [115]. On the other hand, sterols have been involved in the replication of plant pathogens, for example tomato bushy stunt virus (TBSV) [104]. Due to the high diversity of viruses that exploit the cholesterol biosynthetic pathway for replication, this could consider a common requirement. However, replication of viruses may rely on lipids other than cholesterol, as described for

Another major class of lipids that has been related to viral replication are the fatty acids, whose metabolism has been shown to be required for the multiplication of viruses such as brome mosaic virus (BMV) [100,101], Droshophila C virus (DCV) [103], CVB3 [117], and PV [118]. In some cases, in addition to the dependence of cholesterol (discussed in the previous paragraph), viral multiplication is also dependent on fatty acid synthesis. Examples of viruses sharing both cholesterol and fatty acid requirements include DENV [102], WNV [81,102], and HCV [8,112]. Indeed, during DENV infection, the key enzyme responsible for fatty acid synthesis, the fatty acid synthase (FASN), is recruited to the viral replication complex by direct interaction with the viral protein NS3, enhancing its activity [102]. Dependence of DENV replication on fatty acids is shared by mammalian and mosquito host cells [9]. Even more, DENV modulates lipid metabolism through induction of a form of autophagy that targets lipid droplet stores, promoting the depletion of cellular triglycerides and the release of fatty acids. This results on an increase in β-oxidation and ATP production that stimulate viral replication [1,5]. Infection with other viruses (HCV or CVB3) also relies on fatty acids and results in an increase on FASN expression [117,119], a phenomenon that

Besides cholesterol and fatty acids, specific phospholipids can also play a key role in viral replication. For instance, replication of TBSV and FHV is dependent on phospholipid biosynthesis [105]. Replication of FHV was initially associated to glycerophospholipids, being independent of cholesterol or sphingomyelin (a membrane phospholipid that is not derived from glycerol) [116]. However, recent advances on the biology of FHV indicate that

Delta(9) fatty acid desaturase; PI4KIIIα and β, phosphatidylinositol 4-kinase class III α and β; PI4P,

regulatory element binding protein; TBSV, tomato bushy stunt virus; WNV, West Nile virus.

the alphanodavirus flock house virus (FHV) [116].

does not occur upon DENV or WNV infection [81,102].

INO2 Regulation of phospholipid synthesis [105]

The presence of specific lipids in the viral replication complex can accomplish with several missions. For instance, post-translational modification of viral proteins by lipids is associated to viral replication functions [128,129]. Table 3 displays representative examples of lipid functions during viral replication.


Abbreviations used in this table: BMV, brome mosaic virus; HCV, hepatitis C virus; DENV, Dengue virus; FHV, flock house virus; PI4P, phosphatidylinositol 4-phosphate; PV, poliovirus; WNV, West Nile virus.

**Table 3.** Examples of lipid roles during viral replication

Lipids can contribute to viral replication by acting as scaffolding molecules to anchor viral proteins. In PV, location of specific phospholipids (PI4P) to the viral replication sites mediates direct recruitment of the RNA dependent RNA polymerase (the enzyme that replicates the viral genome), which specifically interacts with this lipid [93]. The RNA polymerase of FHV also interacts with a specific phospholipid, the cardiolipin located on the outer mitochondrial membrane, where its replication takes place [120]. In addition to these examples, different events related to the replication of viral genomes are also influenced by specific phospholipids [131,132]. The activation of HCV replication due to a direct binding of sphingomyelin to HCV RNA polymerase has also been documented [130].

Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 301

**Figure 3.** Virus budding through the plasma membrane. Images correspond to BHK-21 cells infected with the rhabdovirus VSV, fixed, and processed for transmission electron microscopy at 7 h postinfection. For technical details related to virus infection and sample preparation see [145].

of DENV and HCV [78,79,146]. Assembly of HCV particles occurs on the surface of lipid droplets and, as mentioned earlier, it is related to the very low density lipoprotein (VLDL) pathway, a phenomenon that leads to the formation of unique lipo-viro-particles [4,147]. The lipoprotein component associated to HCV particles is essential for their infectivity [148], since one of its functions is to interact with LDL-R, thus contributing to viral attachment

Lipids incorporated to viral proteins due to post-translational modifications are also involved in crucial steps of enveloped virus assembly [149,150]. Regarding morphogenesis of non-enveloped viruses, myristoylation of VP4 protein of PV and FMDV has been shown

Differences on the lipid composition of the viral membranes may reflect their different origin. Despite that the lipid content of enveloped viruses has been studied for decades [153- 155], quantitative analyses of viral lipidomes (the entire content of lipids) at the individual molecular species level have not been possible until recently, by means of the improvement of mass spectrometry [3,139]. Following this approach, several studies have extended the knowledge on viral lipid composition. Nevertheless, drawbacks associated to the purification of cellular membranes, in particular the plasma membrane, still complicate the

As commented above, viral membranes can be originated from varied cellular sources (section 2.3). The lipid composition of both Semliki Forest virus (SFV) and VSV is indistinguishable and only displays slight differences with that of the plasma membrane [139]. Being SFV and VSV from different viral families (*Togaviridae* and *Rhabdoviridae*, respectively), these viruses constitute an example of little selection of the lipids included in their envelopes. Since the composition of the viral envelopes of both viruses is similar to that of the plasma membrane, the small differences observed between plasma membrane and these viral envelopes could be explained by the enrichment in specific lipids to facilitate the

In the case of retroviruses (i.e. HIV and murine leukaemia virus), the overall lipid composition of viral envelopes resembles that of detergent-resistant membrane

(section 2.1.1).

necessary for proper capsid assembly [151,152].

*2.3.1. Lipid composition of enveloped viruses* 

analysis of lipid sorting during viral budding [3].

membrane curvature required for viral budding [139].

Proper topology of viral replication complexes usually depends on the induction of a membrane curvature, which may require the presence of specific proteins [133]. Membrane curvature can also be induced by modification of its lipid structure, either through changes in the polar head group or in the acyl chain composition [2,134]. Thus, during BMV infection, the function of an allele of delta9 fatty acid desaturase, an enzyme that introduces double bond in unsaturated fatty acids, has been associated to viral replication complex assembly to increase membrane fluidity and plasticity [100,101]. The accumulation of coneshaped lipids, such as lysophospholipids, which contain single acyl chain per phospholipid molecule, and of special lipids like cholesterol or cardiolipin, has been associated with alterations on the membrane curvature and plasticity that can contribute to replication complex assembly [1,2,135].

As commented before, the membrane rearrangements resulting from replication complex assembly can also contribute to evade the cellular immune response by hiding viral components from pathogen sensors of the innate immune machinery. Thus, WNV-induced redistribution of cellular cholesterol contributes to down regulate the interferon-stimulated antiviral signalling response to infection [90].

Finally, the reorganization of cellular lipid metabolism during infection can also contribute to the generation of ATP in order to provide energy to support robust viral replication [1,5].

#### **2.3. Lipids and viral morphogenesis**

Most enveloped-viruses acquire their lipid membrane by budding through a cellular membrane that can be provided by different sources. For instance, flaviviruses (i.e. DENV or WNV) bud into the endoplasmic reticulum for acquisition of their envelope [80,82], while VSV (Figure 3), influenza, or HIV acquire their envelope by budding from plasma membrane [136-139]. In other cases, different cellular organelles can contribute with distinct membranes to virus envelopment, is reported for herpersvirus and poxvirus [140-142]. Viruses can take advantage of specific parts of the membrane for their assembly. Cholesterol and lipid raft microdomains play an important role on the assembly of a variety of viruses [136-138,143]. In HIV, the presence of PI(4,5)P2 on the membrane is also necessary for assembly and budding of viral particles, and the viral protein Gag localizes to assembly sites via the interaction with this lipid [144]. The synthesis of fatty acids has also been associated to the envelopment of viruses [6].

Other cellular lipid structures play a role on the assembly of a number of viruses. Thus, of intracellular lipid droplets have been associated with the assembly and morphogenesis

**Figure 3.** Virus budding through the plasma membrane. Images correspond to BHK-21 cells infected with the rhabdovirus VSV, fixed, and processed for transmission electron microscopy at 7 h postinfection. For technical details related to virus infection and sample preparation see [145].

of DENV and HCV [78,79,146]. Assembly of HCV particles occurs on the surface of lipid droplets and, as mentioned earlier, it is related to the very low density lipoprotein (VLDL) pathway, a phenomenon that leads to the formation of unique lipo-viro-particles [4,147]. The lipoprotein component associated to HCV particles is essential for their infectivity [148], since one of its functions is to interact with LDL-R, thus contributing to viral attachment (section 2.1.1).

Lipids incorporated to viral proteins due to post-translational modifications are also involved in crucial steps of enveloped virus assembly [149,150]. Regarding morphogenesis of non-enveloped viruses, myristoylation of VP4 protein of PV and FMDV has been shown necessary for proper capsid assembly [151,152].

#### *2.3.1. Lipid composition of enveloped viruses*

300 Lipid Metabolism

complex assembly [1,2,135].

antiviral signalling response to infection [90].

**2.3. Lipids and viral morphogenesis** 

to the envelopment of viruses [6].

mediates direct recruitment of the RNA dependent RNA polymerase (the enzyme that replicates the viral genome), which specifically interacts with this lipid [93]. The RNA polymerase of FHV also interacts with a specific phospholipid, the cardiolipin located on the outer mitochondrial membrane, where its replication takes place [120]. In addition to these examples, different events related to the replication of viral genomes are also influenced by specific phospholipids [131,132]. The activation of HCV replication due to a direct binding

Proper topology of viral replication complexes usually depends on the induction of a membrane curvature, which may require the presence of specific proteins [133]. Membrane curvature can also be induced by modification of its lipid structure, either through changes in the polar head group or in the acyl chain composition [2,134]. Thus, during BMV infection, the function of an allele of delta9 fatty acid desaturase, an enzyme that introduces double bond in unsaturated fatty acids, has been associated to viral replication complex assembly to increase membrane fluidity and plasticity [100,101]. The accumulation of coneshaped lipids, such as lysophospholipids, which contain single acyl chain per phospholipid molecule, and of special lipids like cholesterol or cardiolipin, has been associated with alterations on the membrane curvature and plasticity that can contribute to replication

As commented before, the membrane rearrangements resulting from replication complex assembly can also contribute to evade the cellular immune response by hiding viral components from pathogen sensors of the innate immune machinery. Thus, WNV-induced redistribution of cellular cholesterol contributes to down regulate the interferon-stimulated

Finally, the reorganization of cellular lipid metabolism during infection can also contribute to the generation of ATP in order to provide energy to support robust viral replication [1,5].

Most enveloped-viruses acquire their lipid membrane by budding through a cellular membrane that can be provided by different sources. For instance, flaviviruses (i.e. DENV or WNV) bud into the endoplasmic reticulum for acquisition of their envelope [80,82], while VSV (Figure 3), influenza, or HIV acquire their envelope by budding from plasma membrane [136-139]. In other cases, different cellular organelles can contribute with distinct membranes to virus envelopment, is reported for herpersvirus and poxvirus [140-142]. Viruses can take advantage of specific parts of the membrane for their assembly. Cholesterol and lipid raft microdomains play an important role on the assembly of a variety of viruses [136-138,143]. In HIV, the presence of PI(4,5)P2 on the membrane is also necessary for assembly and budding of viral particles, and the viral protein Gag localizes to assembly sites via the interaction with this lipid [144]. The synthesis of fatty acids has also been associated

Other cellular lipid structures play a role on the assembly of a number of viruses. Thus, of intracellular lipid droplets have been associated with the assembly and morphogenesis

of sphingomyelin to HCV RNA polymerase has also been documented [130].

Differences on the lipid composition of the viral membranes may reflect their different origin. Despite that the lipid content of enveloped viruses has been studied for decades [153- 155], quantitative analyses of viral lipidomes (the entire content of lipids) at the individual molecular species level have not been possible until recently, by means of the improvement of mass spectrometry [3,139]. Following this approach, several studies have extended the knowledge on viral lipid composition. Nevertheless, drawbacks associated to the purification of cellular membranes, in particular the plasma membrane, still complicate the analysis of lipid sorting during viral budding [3].

As commented above, viral membranes can be originated from varied cellular sources (section 2.3). The lipid composition of both Semliki Forest virus (SFV) and VSV is indistinguishable and only displays slight differences with that of the plasma membrane [139]. Being SFV and VSV from different viral families (*Togaviridae* and *Rhabdoviridae*, respectively), these viruses constitute an example of little selection of the lipids included in their envelopes. Since the composition of the viral envelopes of both viruses is similar to that of the plasma membrane, the small differences observed between plasma membrane and these viral envelopes could be explained by the enrichment in specific lipids to facilitate the membrane curvature required for viral budding [139].

In the case of retroviruses (i.e. HIV and murine leukaemia virus), the overall lipid composition of viral envelopes resembles that of detergent-resistant membrane microdomains [156,157]. An enrichment in PI(4,5)P2 has also been documented in HIV envelope, which is compatible with the dependence on the interaction between the viral protein Gag and this lipid to promote HIV budding from plasma membrane [157]. Another virus that buds from membrane rafts is influenza virus [138]. The lipidome of this virus has been analyzed for viruses budding from the apical membrane of polarized cells [158]. This study revealed that that the apical cellular membrane was enriched in sphingolipids and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. These results are consistent with an accumulation of lipid rafts at the membranes where the virus buds. In addition, the virus membrane exhibited a further enrichment of sphingolipids and cholesterol when compared with the donor membrane at the expense of phosphatidylcholines [158].

Lipid Involvement in Viral Infections:

[145]

Present and Future Perspectives for the Design of Antiviral Strategies 303

development of viral resistance than strategies focused on viral proteins. Another advantage of this approach is that compounds targeting a specific group of lipids can successfully inhibit replication of different unrelated viruses (Table 4), thus constituting candidates for broad-spectrum antiviral drugs. These facts make that the use of drugs that impair different aspects of lipid metabolism has been proposed as a feasible antiviral approach [1,6,14].

U18666A DENV, HCV [113,177]

Arachidonate HCV [178] Oleic acid PV [118]

Abbreviations used in this table: AiV, Aichi virus; ASFV, African swine fever virus; CVB, coxsackievirus B; C75, trans-4-carboxy-5-octyl-3-methylene-butyrolactone; DENV, Dengue virus; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; LCMV, lymphocytic chioriomeningitis virus; PV, poliovirus; SINV, Sindbis virus; SFV, Semliki Forest virus; TOFA, 5-tetradecyloxy-2-furoic acid; USUV, Usutu virus; VACV, vaccinia

Cholesterol is involved in multiple steps of the viral cycle. Impairment of cholesterol biosynthetic pathway by inhibitors of 3-hydroxy-3-methyl-glutaryl-CoaA reductase (3- HMG-CoA reductase) like statins, commonly used in treatment of cardiovascular disease, constitutes a novel antiviral approach [174,175,183]. The clinical success of these inhibitors for human disorders also indicates that inhibitors of lipid metabolism can be safe and effective for human therapy. An additional effect of the treatment with statins, unrelated to the inhibition of 3-HMG-CoA reductase, is the inhibition of the binding of leukocyte function-associated antigen-1 (LFA-1) to the intercellular adhesion molecule (ICAM-1) [184], thus being immunomodulators and anti-inflammatory agents [185,186]. These properties

The infection with the paramyxovirus respiratory syncitial virus (RSV) is dependent on the isoprenylation at the carboxy terminus of the cellular protein RhoA by geranylgeranyltransferase. Lovastatin, which blocks prenylation pathways in the cell by directly inhibiting 3-HMG-CoA reductase, inhibits RSV infection both in cultured cells and in mice [183]. Treatment of patients with different statins (i.e. lovastatin, simvastin, or

PIK93 PV, CVB3, CVB5 [81,93,109]

C75 HCMV, DENV, YFV, WNV, Influenza, HCV, CVB3 [6,9,81,102,117,119] Cerulenin DENV, WNV, PV, CVB3 [81,102,117,121]

PV, AiV [108,109]

**Target lipid Inhibitor Antiviral activity against Refs.**  Cholesterol Statins HIV, HCV, influenza [170-176]

Fatty acids TOFA HCMV, Influenza [6]

 AL-9 HCV [179] Sphingolipids Myriocin Hepatitis B virus, HCV [180-182]

Multiple Valproic acid VACV, WNV, SFV, SINV, ASFV, VSV, LCMV,

**Table 4.** Examples of drugs targeting lipid metabolism with reported antiviral activity

USUV

virus; VSV, vesicular stomatitis virus; WNV, West Nile virus; YFV, yellow fever virus

**3.1. Targeting cholesterol as an antiviral strategy** 

can carry additional advantages for fighting HIV [174-176].

PI4P Enviroximelike

In other cases, major differences in lipid content between viral envelopes and host cell membranes have been found. An interesting example is the envelope of HCMV, which contains more phosphatidylethanolamines and less phosphatidylserines than the host cell membranes, resembling the synaptic vesicle lipidome [159]. Another virus with marked differences with cellular membranes is HCV, whose particles show a unique lipid composition in comparison with all other viruses analyzed to date. In addition, the lipid content of the HCV envelope is also different from that of the cells in which it was produced (cholesteryl esters comprise almost half of the total HCV lipids), resembling the composition of VLDL and LDL [160]. This finding is compatible with the association of HCV assembly with the VLDL pathway that leads to the formation of lipo-viro-particles [4,147].
