**3. Targeting lipid metabolism, a novel antiviral strategy**

Specific lipids are essential for multiple steps of the viral replication cycle and, therefore, different strategies can be used to interfere with virus infection. As a first approach to inhibit enveloped virus multiplication, the functions of lipids incorporated into the viral particle can be targeted by chemical compounds or even by antibodies [161]. This is the case of broad-spectrum antivirals, − some of them already licensed for human use, such as arbidol [162-164] −, or inhibitors of membrane fusion [3]. Impairment of viral fusion can be achieved also by targeting viral machinery involved in this process, a strategy currently assayed for HIV treatment [165].

An alternative, non-excluding lipid-targeted strategy to prevent viral multiplication is based on inhibitors of enzymes that catalyse lipid metabolic fluxes upregulated by viral infections [6]. Examples of compounds that act at distinct points of lipid metabolism and with reported antiviral activity *in vitro* are given in Table 4. Targeting lipid metabolism as an antiviral strategy raises important concerns. On one hand, alteration of such important metabolic pathway for cellular homeostasis may resemble a non-specific strategy, which could result in deleterious effects for the host. However, it should be also considered that currently antiviral compounds also target other major metabolic pathways, i.e. that of nucleic acids metabolism [166-169]. On the other hand, targeting host factors to avoid viral replication could also carry advantages. Drugs that target host factors seem to be less susceptible to the 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].


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

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

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

302 Lipid Metabolism

HIV treatment [165].

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

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

Specific lipids are essential for multiple steps of the viral replication cycle and, therefore, different strategies can be used to interfere with virus infection. As a first approach to inhibit enveloped virus multiplication, the functions of lipids incorporated into the viral particle can be targeted by chemical compounds or even by antibodies [161]. This is the case of broad-spectrum antivirals, − some of them already licensed for human use, such as arbidol [162-164] −, or inhibitors of membrane fusion [3]. Impairment of viral fusion can be achieved also by targeting viral machinery involved in this process, a strategy currently assayed for

An alternative, non-excluding lipid-targeted strategy to prevent viral multiplication is based on inhibitors of enzymes that catalyse lipid metabolic fluxes upregulated by viral infections [6]. Examples of compounds that act at distinct points of lipid metabolism and with reported antiviral activity *in vitro* are given in Table 4. Targeting lipid metabolism as an antiviral strategy raises important concerns. On one hand, alteration of such important metabolic pathway for cellular homeostasis may resemble a non-specific strategy, which could result in deleterious effects for the host. However, it should be also considered that currently antiviral compounds also target other major metabolic pathways, i.e. that of nucleic acids metabolism [166-169]. On the other hand, targeting host factors to avoid viral replication could also carry advantages. Drugs that target host factors seem to be less susceptible to the

with the donor membrane at the expense of phosphatidylcholines [158].

with the VLDL pathway that leads to the formation of lipo-viro-particles [4,147].

**3. Targeting lipid metabolism, a novel antiviral strategy** 

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

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 fluvastin) resulted in diverse effects on HCV infection, ranging from an absence of antiviral effect to a modest improvement of sustained antiviral response, or a reduction of viremia [172,173,187-189]. Beneficial effects derived from treatment with statins of infection with diverse influenza strains have also been reported in animal models and human studies [170,171], although other studies do not support these conclusions [190].

Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 305

[102]. Inhibition of this interaction could contribute to the design of antivirals to fight this

Infection of HCV is intimately connected to lipid metabolism, including the fatty acid biosynthetic pathway [4,8], and its replication can be inhibited by C75 [119]. Indeed, fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation [112]. Arachidonate, a polyunsaturated fatty acid, also inhibits HCV replication [112] via the lipid peroxidation induced by reactive oxygen species (ROS) derived from HCV replication that converts polyunsaturated fatty acids into reactive carbonyls that inactivate proteins [178]. These events can be prevented by treatment with the antioxidant vitamin E [178]. As a result of the connections between fatty acids and cholesterol biosynthetic pathways, inhibition of fatty acid synthesis can be also related to the reduction of the infection of HCV, through inhibition of the geranylgeranylation of cellular factors required for HCV replication [112,196]. The use of unsaturated fatty acids has also been

applied to block myristoylation of HIV Gag protein to prevent virus budding [150].

Viral replication also relies on phospholipid biosynthesis [105,121]. This makes drugs interfering this pathway candidates for antiviral design. Along this line, the antiviral properties of valproic acid - a short chain fatty acid commonly used for the treatment of epilepsy and bipolar disorder that impairs multiple aspects of phosphoinositide metabolism [197-199] - against a broad panel of enveloped viruses have been reported (Table 4) [145].

Treatment with a chimeric antibody directed against phospahtidylserine can cure arenavirus and cytomegalovirus infections in animal models [200]. The mechanism of action of this therapy is based on the exposure of phosphatidylserine on the external leaflet of the plasma membrane, a preapoptotic event in cells infected by a broad variety of viruses. The safety and pharmacokinetics of this antibody have been already evaluated in clinical trials for treatment of other human disorders [201]. Another example of the use of antiphospholipidantibodies to combat a viral disease is provided by HIV, since different antiphospholipid antibodies have shown a broad neutralizing activity against this virus [161].

Recent reports have highlighted the role of a specific phospholipid species, PI4P, in the replication of enteroviruses (PV, CVB3) and HCV (section 2.2.2). The synthesis of PI4P associated to viral replication relies on the function of the cellular enzymes PI4KIIIα and β. This makes both lipid kinases potential drug targets for antiviral design. An inhibitor of PI4KIIIβ, PIK93 [202], has been shown to impair replication of enteroviruses [81,93,109]. Related reports have also uncovered that this enzyme is the cellular target of known antiviral compounds against enteroviruses [109]. This is so for some enviroxime-like compounds − T-00127-HEV1 and GW5074 [93] − that integrate a group of antivirals that inhibits enterovirus replication, for which mutations conferring drug resistance mapped to the same region of the enteroviral protein 3A [109,203-205]. The recruitment of PI4KIIIβ to viral replication complexes requires the participation of cellular partners like the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), as

important human disease.

**3.3. Phosphlipids as antiviral targets** 

Targeting cholesterol in viral infection can be achieved using drugs other than statins, for instance U18666A. This is an intracellular cholesterol transport inhibitor widely used to block the intracellular trafficking of cholesterol and mimic Niemann-Pick type C disease, which also blocks cholesterol biosynthesis by inhibiting oxidosqualene cyclase and desmosterol reductase [191]. Treatment with U18666A inhibits DENV infection in cultured cells, and the effect of this compound is additive to the inhibitory effect of C75 (an inhibitor of FASN), which shows that both, cholesterol and fatty acids, are required for successful DENV replication [177]. U18666A also displays an antiviral effect against HCV infection and a synergistic effect has been reported when combined with interferon [113].

### **3.2. Inhibitors of fatty acid biosynthesis as potential antiviral compounds**

The biosynthesis of fatty acids plays an important role for multiplication of a wide variety of viruses [6,9,81,102]. Pharmacological inhibition of this metabolic pathway can be achieved using 5-tetradecyloxy-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase (ACC) [192]. Treatment with this compound has been shown to block replication of HCMV and influenza A virus [6]. Although the results derived from these experiments performed in model cell culture systems need to be further reproduced using animal models, the concentrations of TOFA that successfully inhibit HCMV infection in cultured cells are in the range of plasma concentrations found in rats treated with this inhibitor [6]. In HCV, treatment with TOFA attenuates the enhancement of replication of HCV induced by ethanol [193].

On the other hand, treatment with trans-4-carboxy-5-octyl-3-methylene-butyrolactone (C75) − an inhibitor of FASN, the key enzyme of fatty acid biosynthetic pathway − also resulted in inhibition of the replication of both HCMV and influenza A virus [6]. These experiments were performed in cultured cells, and the concentrations of the inhibitor tested did not induce host cell toxicity or apoptosis [6]. Similar results have been obtained for DENV, yellow fever virus (YFV), WNV, and HCV using the FASN inhibitor C75 [9,81,102,119], or cerulenin, another FASN inhibitor [81,102,111]. HMCV, influenza A, DENV, YFV, and WNV are enveloped viruses. The antiviral effect of either cerulenin or C75 has also been probed for the non-enveloped viruses CVB3 and PV [117,121], enlarging the potential antiviral spectrum of FASN inhibitors. However, it should be noted that blockage of FASN by C75 can cause severe anorexia and weight loss in animal models [194]. This makes of C75 a drug not aimed for human use, although it has aided to the identification of potential pathways to target obesity [195], and is also contributing to the understanding of the relationship between biosynthetic fatty acid synthesis and viral multiplication [6,9,81,102,117]. For DENV, a direct interaction between the viral protein NS3 and FASN has been reported [102]. Inhibition of this interaction could contribute to the design of antivirals to fight this important human disease.

Infection of HCV is intimately connected to lipid metabolism, including the fatty acid biosynthetic pathway [4,8], and its replication can be inhibited by C75 [119]. Indeed, fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation [112]. Arachidonate, a polyunsaturated fatty acid, also inhibits HCV replication [112] via the lipid peroxidation induced by reactive oxygen species (ROS) derived from HCV replication that converts polyunsaturated fatty acids into reactive carbonyls that inactivate proteins [178]. These events can be prevented by treatment with the antioxidant vitamin E [178]. As a result of the connections between fatty acids and cholesterol biosynthetic pathways, inhibition of fatty acid synthesis can be also related to the reduction of the infection of HCV, through inhibition of the geranylgeranylation of cellular factors required for HCV replication [112,196]. The use of unsaturated fatty acids has also been applied to block myristoylation of HIV Gag protein to prevent virus budding [150].

#### **3.3. Phosphlipids as antiviral targets**

304 Lipid Metabolism

[193].

fluvastin) resulted in diverse effects on HCV infection, ranging from an absence of antiviral effect to a modest improvement of sustained antiviral response, or a reduction of viremia [172,173,187-189]. Beneficial effects derived from treatment with statins of infection with diverse influenza strains have also been reported in animal models and human studies

Targeting cholesterol in viral infection can be achieved using drugs other than statins, for instance U18666A. This is an intracellular cholesterol transport inhibitor widely used to block the intracellular trafficking of cholesterol and mimic Niemann-Pick type C disease, which also blocks cholesterol biosynthesis by inhibiting oxidosqualene cyclase and desmosterol reductase [191]. Treatment with U18666A inhibits DENV infection in cultured cells, and the effect of this compound is additive to the inhibitory effect of C75 (an inhibitor of FASN), which shows that both, cholesterol and fatty acids, are required for successful DENV replication [177]. U18666A also displays an antiviral effect against HCV infection and

[170,171], although other studies do not support these conclusions [190].

a synergistic effect has been reported when combined with interferon [113].

**3.2. Inhibitors of fatty acid biosynthesis as potential antiviral compounds** 

The biosynthesis of fatty acids plays an important role for multiplication of a wide variety of viruses [6,9,81,102]. Pharmacological inhibition of this metabolic pathway can be achieved using 5-tetradecyloxy-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase (ACC) [192]. Treatment with this compound has been shown to block replication of HCMV and influenza A virus [6]. Although the results derived from these experiments performed in model cell culture systems need to be further reproduced using animal models, the concentrations of TOFA that successfully inhibit HCMV infection in cultured cells are in the range of plasma concentrations found in rats treated with this inhibitor [6]. In HCV, treatment with TOFA attenuates the enhancement of replication of HCV induced by ethanol

On the other hand, treatment with trans-4-carboxy-5-octyl-3-methylene-butyrolactone (C75) − an inhibitor of FASN, the key enzyme of fatty acid biosynthetic pathway − also resulted in inhibition of the replication of both HCMV and influenza A virus [6]. These experiments were performed in cultured cells, and the concentrations of the inhibitor tested did not induce host cell toxicity or apoptosis [6]. Similar results have been obtained for DENV, yellow fever virus (YFV), WNV, and HCV using the FASN inhibitor C75 [9,81,102,119], or cerulenin, another FASN inhibitor [81,102,111]. HMCV, influenza A, DENV, YFV, and WNV are enveloped viruses. The antiviral effect of either cerulenin or C75 has also been probed for the non-enveloped viruses CVB3 and PV [117,121], enlarging the potential antiviral spectrum of FASN inhibitors. However, it should be noted that blockage of FASN by C75 can cause severe anorexia and weight loss in animal models [194]. This makes of C75 a drug not aimed for human use, although it has aided to the identification of potential pathways to target obesity [195], and is also contributing to the understanding of the relationship between biosynthetic fatty acid synthesis and viral multiplication [6,9,81,102,117]. For DENV, a direct interaction between the viral protein NS3 and FASN has been reported Viral replication also relies on phospholipid biosynthesis [105,121]. This makes drugs interfering this pathway candidates for antiviral design. Along this line, the antiviral properties of valproic acid - a short chain fatty acid commonly used for the treatment of epilepsy and bipolar disorder that impairs multiple aspects of phosphoinositide metabolism [197-199] - against a broad panel of enveloped viruses have been reported (Table 4) [145].

Treatment with a chimeric antibody directed against phospahtidylserine can cure arenavirus and cytomegalovirus infections in animal models [200]. The mechanism of action of this therapy is based on the exposure of phosphatidylserine on the external leaflet of the plasma membrane, a preapoptotic event in cells infected by a broad variety of viruses. The safety and pharmacokinetics of this antibody have been already evaluated in clinical trials for treatment of other human disorders [201]. Another example of the use of antiphospholipidantibodies to combat a viral disease is provided by HIV, since different antiphospholipid antibodies have shown a broad neutralizing activity against this virus [161].

Recent reports have highlighted the role of a specific phospholipid species, PI4P, in the replication of enteroviruses (PV, CVB3) and HCV (section 2.2.2). The synthesis of PI4P associated to viral replication relies on the function of the cellular enzymes PI4KIIIα and β. This makes both lipid kinases potential drug targets for antiviral design. An inhibitor of PI4KIIIβ, PIK93 [202], has been shown to impair replication of enteroviruses [81,93,109]. Related reports have also uncovered that this enzyme is the cellular target of known antiviral compounds against enteroviruses [109]. This is so for some enviroxime-like compounds − T-00127-HEV1 and GW5074 [93] − that integrate a group of antivirals that inhibits enterovirus replication, for which mutations conferring drug resistance mapped to the same region of the enteroviral protein 3A [109,203-205]. The recruitment of PI4KIIIβ to viral replication complexes requires the participation of cellular partners like the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), as

described for AiV [108,122]. In this way, modulation of the interaction between these proteins could also constitute a novel antiviral strategy [122].

Lipid Involvement in Viral Infections:

Present and Future Perspectives for the Design of Antiviral Strategies 307

metabolism. Indeed, great progresses have been recently made in this area due to the identification of specific lipids as key factors for viral multiplication. However, the specific function of most of these lipids remains to be determined. A better understanding of the interactions between viral infections and lipid metabolism is desirable to asses the roles of lipids in viral multiplication. This knowledge could lead to the identification of lipid targets and druggable metabolic pathways suitable for antiviral development. Lipid candidates for these interventions have already been identified, for instance fatty acids, cholesterol or specific phospholipids. Indeed, initial lipid-based antiviral approaches have been already started, even at clinical level (i.e. statins). This example has probed that drugs already licensed for humans that act at different points of lipid metabolism can constitute potential candidates to fight viral diseases. These lipid-targeted antiviral approaches could be exclusive or could also be complementary to other antiviral therapies already available.

As recently remarked, 'if RNA ruled the last decade and DNA dominated the previous one, could the next decade be the one for lipids?' [208]. The new advances on the knowledge of the interplay between viruses and lipids evidence that the answer to this question could be 'Yes' in the case of virology [1]. Hopefully, we are now assisting to the promising birth of a novel lipid-based branch of antiviral research focused on this challenging and still poorly

Miguel A. Martín-Acebes, Ángela Vázquez-Calvo, Flavia Caridi and Francisco Sobrino *Department of Virology and Microbiology, Centre for Molecular Biology "Severo Ochoa" (CBMSO)* 

*Department of Biotechnology, National Institute for Food Science and Technology (INIA), Madrid,* 

Work at CBMSO was supported by grants from Ministerio de Ciencia e Innovación (MICINN, Spain) BIO2008-0447-C03-01, and BIO2011-24351, and by an institutional grant

Work at INIA was supported by grants RTA2011-00036 from the Spanish Ministerio de Ciencia e Innovación (MICINN) and the Network of Animal Disease Infectiology and Research-European Union NADIR-EU-228394. MAMA is the recipient of a JAE-Doc

[1] Heaton NS, Randall G (2011) Multifaceted roles for lipids in viral infection. Trends

explored field for drug discovery.

*(UAM/CSIC), Cantoblanco, Madrid, Spain* 

from the "Fundación Ramón Areces".

fellowship from Spanish Research Council (CSIC).

**Author details** 

Juan-Carlos Saiz

**5. References** 

Microbiol 19: 368-375.

**Acknowledgement** 

*Spain* 

In HCV infection the isoform of the enzyme involved in viral replication is mainly PI4KIIIα [106,107], although a role of the β isoform in the inhibition of HCV replication due to treatment with PIK93 has also been reported [126]. Consistent with these findings, specific compounds that successfully inhibit PV replication through blockage of PI4KIIIβ (GW5074 and T-00127-HEV1) do not affect HCV replication, although other enviroxime-like compounds can affect both enteroviral and HCV replication [109]. On the other hand, 4 anilino quinazolines were first reported to have antiviral activity against HCV, although the mechanism was not well defined. However, a recent study has associated the antiviral activity of a representative 4-anilino quinazoline (AL-9) with the inhibition of PI4KIIIα during HCV infection, which opens new therapeutic approaches [179]. Since the viral protein NS5A directly interacts with PI4KIIIα during HCV infection [125,127], modulation of this interaction also raises novel possibilities for antiviral research.

Overall, these examples of drugs targeting different enzymes related to phosphoinositide metabolism support this strategy as a feasible approach for antiviral drug discovery. In this line, related phosphoinositide kinases constitute an important emerging class of drug targets [202].

#### **3.4. Sphingolipids as antiviral targets**

Sphingolipids constitute a major component of lipid rafts [37,38], which, as commented before, are involved in different steps of viral infection, making sphingolipids potential antiviral targets. Along this line, ebolavirus requires the activity of acid sphingomyelinase, the enzyme that converts sphingomyelin to phosphocholine and ceramide for infection, and depletion of sphingomyelin reduces its infection [206]. A dependence on sphingomyelin for HCV replication has also been documented [130]. Inhibition of serine palmitoyltransferase, the enzyme that catalyzes the first step on sphingolipid biosynthesis, using myriocin has also been assayed against HCV or hepatitis B virus, either alone or in combination with interferon [180-182]. However, a certain controversy exists regarding whether the inhibitory effect of myriocin on HCV replication is attributable to the specific inhibition of serine palmitoyltransferase, since FTY720, a compound that like myriocin is structurally similar to sphingosine but does not inhibit serine palmitoyltransferase, also inhibits HCV replication [207].

#### **4. Conclusion**

The analysis of the functions of cellular factors in viral infections has highlighted the role of different lipid species in these infections. Viruses can use cellular lipids like bricks to build viral particles or to develop viral replication complexes, thus facilitating its multiplication. But viruses can also manipulate host cell metabolism towards the production of specific lipid species, unveiling an intimate relationship between viruses and host cell lipid metabolism. Indeed, great progresses have been recently made in this area due to the identification of specific lipids as key factors for viral multiplication. However, the specific function of most of these lipids remains to be determined. A better understanding of the interactions between viral infections and lipid metabolism is desirable to asses the roles of lipids in viral multiplication. This knowledge could lead to the identification of lipid targets and druggable metabolic pathways suitable for antiviral development. Lipid candidates for these interventions have already been identified, for instance fatty acids, cholesterol or specific phospholipids. Indeed, initial lipid-based antiviral approaches have been already started, even at clinical level (i.e. statins). This example has probed that drugs already licensed for humans that act at different points of lipid metabolism can constitute potential candidates to fight viral diseases. These lipid-targeted antiviral approaches could be exclusive or could also be complementary to other antiviral therapies already available.

As recently remarked, 'if RNA ruled the last decade and DNA dominated the previous one, could the next decade be the one for lipids?' [208]. The new advances on the knowledge of the interplay between viruses and lipids evidence that the answer to this question could be 'Yes' in the case of virology [1]. Hopefully, we are now assisting to the promising birth of a novel lipid-based branch of antiviral research focused on this challenging and still poorly explored field for drug discovery.
