West Nile Virus Entry Requires Cholesterol-Rich Membrane Microdomains and Is Independent of {alpha}v{beta}3 Integrin

West Nile virus (WNV) has been the leading cause of viral encephalitisin the United States since 1999. The endocytic processes involvedin the internalization of infectious WNV by various cell typesare not well characterized, and the involvement of cholesterol-richmembrane microdomains, or lipid rafts, in the life cycle ofWNV has not been investigated previously. In this study, wefound that the depletion of cellular cholesterol levels by brieftreatment with methyl-β-cyclodextrin resulted in a 100-foldreduction of the titers of infectious WNV released into theculture supernatant, as well as a reduction in the number ofWNV genome copies in the cholesterol-depleted cells. The additionof exogenous cholesterol to cholesterol-depleted cells reversedthis effect. Cholesterol depletion postinfection did not affectWNV growth, suggesting that the effect occurs at the level ofWNV entry. We also showed that while WNV entry did not require ${alpha}$ vβ3 integrin and focal adhesion kinase, WNV particles failedto be internalized by cholesterol-depleted cells. Finally, weshowed the colocalization of the WNV envelope protein and choleratoxin B, which is internalized in a lipid raft-dependent pathway,in microdomain clusters at the plasma membrane. These data suggestthat WNV utilizes lipid rafts during initial stages of internalizationand that the lipid rafts may contain a factor(s) that may enhanceWNV endocytosis.

INTRODUCTION

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INTRODUCTION

West Nile virus (WNV) belongs to the family Flaviviridae andthe genus Flavivirus, which includes pathogens of global concernsuch as dengue virus (DENV), Japanese encephalitis virus, andyellow fever virus. Severe forms of WNV disease manifest asneurological symptoms such as meningitis, encephalitis, andpoliomyelitis. WNV is a single-stranded positive-sense RNA virus.The 11-kb WNV RNA genome encodes a single polyprotein that iscleaved by the host and viral proteases into three structural(capsid [C], membrane [M], and envelope [E]) and seven nonstructural(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (3). WNVentry into the host cells is mediated by clathrin-coated vesicles(6, 19). Virus delivery into acidic endosomes leads to the uncoatingof the virion, releasing viral RNA into the cytoplasm, followedby RNA replication via minus-strand RNA intermediates (3). Themechanism of WNV entry and the relative roles of different endocyticpathways in WNV internalization have not been well characterized.

Lipid rafts are membrane microdomains enriched with cholesteroland glycosphingolipids (10). They perform crucial roles in theestablishment of cell polarity and act as platforms mediatingsignal transduction and protein trafficking. Proteins with posttranslationalmodifications, such as a glycosylphosphatidylinositol anchoror palmitoylation, are associated with lipid rafts and are responsiblefor the functions ascribed to lipid rafts. Some of the raft-associatedproteins include Src family kinases, receptor tyrosine kinases,G protein-coupled receptors, integrins, microtubules, and cytoskeletaland intermediate filament proteins (12). Many viruses utilizeraft-mediated trafficking pathways for entering or exiting cells.While some animal viruses like human immunodeficiency virustype 1, coxsackievirus, simian virus 40, and severe acute respiratorysyndrome coronavirus depend on lipid rafts for binding to andentry into the host, others like rotavirus, Newcastle diseasevirus, influenza virus, and Ebola and Marburg viruses utilizeraft-mediated pathways for assembly and egress (4, 22, 26, 27).The involvement of lipid rafts in the life cycle of WNV hasnot been investigated. A previous study with DENV has indicateda role for lipid rafts in the flaviviral life cycle (29). However,the exact step at which lipid rafts play a role and the mechanisminvolved remain uncharacterized. In the present study, we provideevidence to support the role of lipid rafts in WNV entry. Ourresults provide insights into the endocytic pathway involvedin WNV internalization, which will further our understandingof the cellular factors involved in WNV endocytosis.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Cell culture and virus strains. WHO-certified Vero cells were grown in Dulbecco's modified Eagle'smedium (DMEM) containing 10% Fetalplex serum (Gemini Bio-Products),penicillin, streptomycin, and glutamine. SK-N-MC neuroblastomaand HeLa cells were grown in DMEM containing 10% fetal bovineserum with penicillin, streptomycin, and glutamine. CS-1 hamstermelanoma cells were grown in suspension in RPMI 1640 mediumcontaining 10% fetal bovine serum with penicillin, streptomycin,and glutamine (11, 37). Wild-type and β3 integrin-deficientmouse embryonic fibroblasts (MEFs) were grown as described previously(16, 30). Focal adhesion kinase-deficient (FAK^–/–)and FAK^+/+ cells were grown as described previously (33, 35).The WNV strain (NY385-99) used in this study has been describedbefore (25). The infection of cells with WNV and the determinationof viral titers by plaque assays were performed as describedpreviously (15, 25). Vesicular stomatitis virus (VSV) Indiana(strain ATCC VR-158) was purchased from the ATCC. VSV infectionsand plaque assays were performed in the same manner as describedfor WNV except that VSV plaque assay preparations were fixedand stained 48 h postinfection (hpi).

Cholesterol depletion and repletion. Cells grown in 12-well plates were washed three times with phosphate-bufferedsaline (PBS), and DMEM containing 2% delipidated serum (Biomeda)with or without the following concentrations of methyl-β-cyclodextrin(MBCD; Sigma) was added to the cells: 7.5 mM for 1 h for Verocells, 10 mM for 30 min for HeLa cells, and 5 mM for 30 minfor SK-N-MC cells. Plates were incubated at 37°C on a rockerfor the indicated periods. After treatment, the cells were washedthree times with PBS and infected with WNV in the above-mentionedmedium without MBCD. Cholesterol replenishment was done as describedin earlier studies (7, 9, 28). Briefly, cholesterol was depletedas described above, and cells were washed three times with PBSand incubated with 400 µg of water-soluble cholesterol(Sigma)/ml in the above-mentioned medium with 2 mM MBCD for1 h. Cells were washed again three times with PBS and infectedwith WNV as described above. The level of viability of untreatedor MBCD-treated cells was found to be >90%, as analyzed bytrypan blue exclusion. MBCD treatment did not affect cell proliferation,as evaluated by cell counting before and after a 48-h treatment(data not shown).

Quantitative real-time RT-PCR. Cells were collected in 0.5 to 1 ml of TRIzol reagent (Invitrogen),and RNA was prepared according to the instructions of the reagentmanufacturer. One hundred nanograms of RNA was used for determiningWNV genome copy numbers by analysis with a one-step reversetranscription-PCR (RT-PCR) system (Applied Biosystems) as describedpreviously (25). Genome copy numbers were normalized to GAPDH(glyceraldehyde-3-phosphate dehydrogenase) or β-actin genecopy numbers by using the comparative cycle threshold valuesdetermined in parallel (25). An Applied Biosystems inventoriedassay (assay identification no. Hs00168352_m1) was used to estimatethe copy numbers of 3-hydroxy-methylglutaryl coenzyme A reductase(HMGCR) mRNA at the time points indicated in Fig. 3 by one-stepRT-PCR using RNA isolated from SK-N-MC cells infected with 10PFU of WNV/cell.

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FIG. 3. Cholesterol depletion postinfection does not affect WNV growth. (A) Vero cells were infected at an MOI of 5 PFU of WNV/cell. Six hours postinfection, cells were treated with MBCD as described above, and cell growth continued until the indicated time points. Viral titers were determined by plaque assays as described in Materials and Methods. Results representative of two independent experiments performed in triplicate are shown. Error bars represent SD (n = 3). (B) SK-N-MC cells were infected at an MOI of 10 PFU of WNV/cell, and at the indicated time points, cells were harvested for RNA isolation. Copy numbers of HMGCR mRNA were estimated by TaqMan gene expression analysis primer-probe sets as described in Materials and Methods. β-actin cycle threshold values were used for normalization. Error bars represent SD (n = 6).

Virus attachment. Vero cells (3 x10⁵) were grown in 12-well plates and treatedwith MBCD as described above. After a 1-h treatment with MBCD,cells were shifted onto ice and washed three times with coldPBS. Cells on ice were infected with WNV diluted in cold DMEMcontaining 2% delipidated serum at a multiplicity of infection(MOI) of 10 PFU/cell (200 µl of virus medium/well). Cellswere incubated at 4°C for 1 h with intermittent mixing.After incubation, virus medium was removed and cells were washedthree times with cold PBS. Cells were collected in 500 µlof TRIzol reagent per well for RNA isolation and WNV genomecopy number determination as described above.

Immunofluorescence microscopy. Cells were grown on 12-mm coverslips in 24-well plates. MBCDtreatment and WNV infection were performed as described above.At 24 hpi, cells were washed twice with PBS, fixed in 4% paraformaldehyde(PFA) for 20 min, permeabilized in PBS containing 0.1% TritonX-100 and 0.2% bovine serum albumin, and blocked with normalgoat serum in the same buffer for 10 min. Samples were incubatedwith anti-E antibodies (purified from D1-4G2 hybridomas fromthe ATCC) for 1 h, followed by rhodamine or fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) secondaryantibody (Invitrogen).

CTB and WNV endocytosis. For cholera toxin B (CTB) endocytosis, cells were washed twicewith PBS and incubated for 30 min at 4°C with DMEM containing0.5% bovine serum albumin with or without 10 µg of FITC-conjugatedCTB (Invitrogen)/ml and with or without purified WNV at an MOIof 10 PFU/cell. After attachment at 4°C, cells were transferredto 37°C for 10 min to allow the endocytosis of CTB and WNV.Cells were washed twice with PBS, fixed with PFA, and processedfor immunofluorescence analysis as described above.

In WNV endocytosis experiments, virus attachment was permittedas described above and virus internalization was allowed for30 min. Cells were fixed and stained as described above withmonoclonal anti-E and anti-early endosome antigen 1 (anti-EEA1)antibodies (BD Biosciences), followed by isotype-specific Alexadye-conjugated goat anti-mouse secondary antibodies (Alexa 488-IgG2a[E] and Alexa 594-IgG1 [EEA1], respectively; Invitrogen).

After final washes, coverslips were mounted onto glass slidesin Prolong antifade reagent with DAPI (4',6'-diamidino-2-phenylindole)to stain the nuclei (Invitrogen). Images were acquired witha DeltaVision real-time image station (Applied Precision Instruments),a wide-field deconvolution microscope system using SoftWoRxas the software application. This system has normal band-passfilters and a Nikon CoolSnap HQ camera. Images were acquiredwith the 60x, 1.45-numerical-aperture lens objective at 0.2µm for Z-stacks in red (Alexa 594; excitation wavelength,594 nm; emission wavelength, 617 nm), green (Alexa 488; excitationwavelength, 488 nm; emission wavelength, 538 nm), and blue (DAPI;excitation wavelength, 360 nm; emission wavelength, 460 nm).These stacks were then deconvolved using nine iterations. Allimages were captured under identical settings. Color levelswere adjusted using Adobe Photoshop with the same template filefor all images. For a projection of six stacks in the middle,see Fig. 2. For a single Z-stack image, see Fig. 5 and 6.

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FIG. 2. Cholesterol depletion results in reduced infection and RNA replication. Vero cells grown on coverslips were treated with MBCD and infected with WNV (MOI of 5 PFU/cell). (A) Twenty-four hours postinfection, cells were fixed with PFA and stained with WNV E antibodies, followed by Alexa 594-conjugated secondary antibodies. The nuclei were stained by DAPI. Scale bars, 20 µm. (B) Lysates from parallel samples were processed for Western analysis to detect WNV NS5. Results for β-actin are shown as a loading control. +, present; –, absent. (C) Vero cells were treated with MBCD and infected with WNV as described above, and 22 hpi, RNA was isolated from cells and WNV genome copy numbers were determined by qRT-PCR as described in Materials and Methods. Results representative of three experiments performed with two or more replicates are shown. Error bars represent SD (n = 6).

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FIG. 5. ${alpha}$ vβ3 integrins are not required for WNV entry. (A) Wild-type (WT) and β3 integrin^–/– MEFs were infected with WNV (MOI of 3 PFU/cell) as described in Materials and Methods. WNV titers in the culture supernatants at 20 hpi were determined by plaque assays on Vero cells. Error bars represent SD (n = 6). (B) FAK^+/+ and FAK^–/– cells were infected with WNV (MOI of 3 PFU/cell), and 18 to 24 hpi, WNV titers in the culture supernatants were determined by plaque assays on Vero cells. Error bars represent SD (n = 6). (C) CS-1 cells were infected with WNV (MOI of 3 PFU/cell), and 24 hpi, WNV titers in the culture supernatants were determined by plaque assays on Vero cells. Error bars represent SD (n = 6). Results representative of at least two independent experiments performed with two or more replicates are shown. ND, not detected.

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FIG. 6. Effect of cholesterol depletion on WNV attachment and internalization. (A) Vero cells were treated with MBCD for 1 h, infected on ice at an MOI of 10 PFU/cell, and incubated at 4°C for 1 h as described in Materials and Methods. Cells were collected in TRIzol for RNA isolation and WNV RNA copy number determination. Results from two independent experiments performed in duplicate are shown. Error bars represent SD (n = 4). (B) Vero cells grown on coverslips were infected at an MOI of 10 PFU of WNV/cell and incubated for 30 min on ice. Cells were shifted to 37°C for 30 min, after which they were fixed in 4% PFA and stained with WNV E and EEA1 antibodies, followed by Alexa 488- and Alexa 594-conjugated secondary antibodies. The nuclei were stained by DAPI. RGB overlay images are shown. Arrows indicate WNV particles in EEA1-positive endosomes in mock-treated cells and the sparse peripheral distribution of WNV particles in MBCD-treated cells. UI, uninfected cells; scale bars, 10 µm.

Western blots. Total protein was isolated from the TRIzol organic phase afterRNA isolation per the instructions of the TRIzol manufacturer(Invitrogen). Equal amounts of protein were loaded onto a 10%sodium dodecyl sulfate-polyacrylamide gel, transferred ontopolyvinylidene difluoride (Immobilon-Millipore), and probedwith rabbit polyclonal antibody against NS5 (a kind gift fromIan Lipkin) and monoclonal anti-β-actin antibodies (Sigma),followed by horseradish peroxidase-conjugated anti-rabbit oranti-mouse antibodies (GE Healthcare). Blots were visualizedby using a SuperSignal West Pico chemiluminescent substrateaccording to the protocol of the manufacturer (Pierce).

Statistical analyses. GraphPad Prism software was used for graphical representationsand statistical analyses. P values were obtained from a two-tailed,unpaired Student t test.

RESULTS

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RESULTS

The depletion of cholesterol leads to a decrease in WNV titers in the supernatant. In order to characterize the role of cholesterol-rich membranemicrodomains, or lipid rafts, in the life cycle of WNV, we treatedVero, SK-N-MC, and HeLa cells with MBCD, which is widely usedto sequester cholesterol from the plasma membrane, prior toinfection with WNV and measured viral titers in the culturesupernatant 22 hpi. As shown in Fig. 1A, we observed a 100-foldreduction in the viral titers in various cell types treatedwith MBCD, suggesting that the lipid rafts play an essentialrole in the life cycle of WNV and that this effect is not celltype specific. The concentrations of MBCD and the periods ofMBCD treatment used in our study have been shown to be nontoxicfor many cell lines (39), and we have verified the same by trypanblue exclusion and found that $~$ 90% of the cells were viable underthese conditions (data not shown).

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FIG. 1. Effect of cholesterol depletion on WNV and VSV titers. (A) Vero, SK-N-MC, and HeLa cells were treated with MBCD and infected with WNV (at MOIs of 5 PFU/cell for Vero cells and 10 PFU/cell for SK-N-MC and HeLa cells) as described in Materials and Methods. WNV titers in the culture supernatants at 22 hpi were determined by plaque assays on Vero cells. Error bars represent standard deviations (SD; n = 4, 3, and 5, respectively). *, **, and *** indicate P values of <0.0003, <0.009, and <0.0001, respectively. (B) SK-N-MC and HeLa cells were treated with MBCD and infected with VSV (MOI of 5 PFU/cell) as described in Material and Methods. VSV titers in the culture supernatants at 16 hpi were determined by plaque assays on Vero cells. Error bars represent SD (n = 6).

The acute depletion of cholesterol by MBCD has been shown previouslyto transiently inhibit clathrin-mediated endocytic pathways(31). To rule out the possibility that the reduction in viraltiters was due to a gross disruption in the membrane architectureand the inhibition of all cellular uptake pathways upon cholesteroldepletion, we treated SK-N-MC and HeLa cells with MBCD as describedabove and infected the cells with VSV, which enters cells viaclathrin-dependent endocytosis (36). We measured viral titersin the supernatants 16 hpi. Under our conditions of cholesteroldepletion of these cells, MBCD treatment did not affect VSVtiters (Fig. 1B). These data suggest a specific requirementfor lipid rafts for normal WNV replication.

Cholesterol-depleted cells have reduced levels of WNV RNA. Cholesterol-rich membrane microdomains are present not onlywithin the plasma membrane but also in the membranes of intracellularorganelles and are involved in protein trafficking from theplasma membrane and in the secretory pathway. The depletionof plasma membrane cholesterol by MBCD may cause the depletionof cholesterol stores at the endoplasmic reticulum as the cellattempts to restore a cholesterol balance. Therefore, the disruptionof lipid rafts by MBCD may have an effect on WNV entry, RNAreplication, assembly, or egress. To identify the stage(s) atwhich lipid rafts play a role in the WNV life cycle, we pretreatedVero cells with MBCD and infected them with WNV as describedabove. Twenty-four hours postinfection, cells were fixed andanalyzed by immunofluorescence with anti-E antibodies. Simultaneously,lysates from mock- or WNV-infected cells were prepared for Westernblot analysis. As shown in Fig. 2A, MBCD-treated cell sampleshad fewer infected cells than mock-treated samples, as seenby staining with WNV E antibody. Western blot analysis of thelysates also showed smaller amounts of WNV NS5 protein in MBCD-treatedcells than in mock-treated cells (Fig. 2B). Similar resultswere obtained for SK-N-MC cells (data not shown). We next determinedthe WNV RNA copy numbers in mock- or MBCD-treated, WNV-infectedcells 24 hpi by quantitative RT-PCR (qRT-PCR). As shown in Fig.2C, we found 100-fold-lower levels of WNV-RNA copies in cellstreated with MBCD than in the mock-treated cells. These resultsindicated that lipid rafts either play a direct role in RNAreplication or function at an earlier stage in the virus lifecycle, possibly during virus entry.

Cholesterol depletion postinfection does not affect WNV titers. A recent study has indicated a role for cholesterol and thecholesterol biosynthetic pathway in the replication of the RNAof Kunjin virus (a nonpathogenic Australian variant of WNV)(21). We determined if cholesterol depletion affects postentryevents in the WNV life cycle by infecting cells with WNV andtreating them with MBCD postinfection. Cells were treated withMBCD 6 hpi as described above, and the viral titers in the culturesupernatants were determined by plaque assays at 6 and 16 hpost-MBCD treatment (12 and 22 hpi, respectively). As shownin Fig. 3A, cholesterol depletion by MBCD postinfection hadno effect on viral titers. Similar results were obtained whencells were treated with MBCD 3 hpi (data not shown), suggestingthat the effect of MBCD is at a step prior to WNV RNA replication.Since it is impossible to completely deplete cellular cholesterolwithout affecting viability, it is possible that the residualcholesterol present in the cells was sufficient to participatein WNV replication. Alternatively, it is possible that cellularcholesterol levels were replenished by de novo synthesis uponthe removal of the drug, allowing for cholesterol participationin viral RNA replication, assembly, or egress.

To determine if cholesterol biosynthesis is upregulated duringWNV infection, SK-N-MC cells were infected with WNV and cellswere collected 8, 24, and 48 hpi for RNA isolation. The levelsof mRNA for HMGCR, an enzyme involved in cholesterol biosynthesis,were measured by qRT-PCR. We saw a modest induction of HMGCRin WNV-infected cells at early time points, and by 48 h, HMGCRlevels were similar to those in uninfected controls (Fig. 3B).In agreement with our results, Mackenzie et al. also observedmuch less induction of HMGCR in cells infected with WNV strainNY99 than in those infected with Kunjin virus (21). This findingsuggests that, unlike the Kunjin virus, WNV does not significantlyinduce cholesterol biosynthesis and that de novo synthesis ofcholesterol may not be required for WNV RNA replication.

WNV entry is dependent on lipid rafts and independent of ${alpha}$ vβ3 integrin. We next tested directly if cholesterol depletion by MBCD affectsWNV entry. Vero cells were pretreated with MBCD and infectedwith WNV as before. After 1 h of incubation at 37°C, cellswere treated with trypsin to release any attached viral particles(see the supplemental material) that failed to be internalizedand washed extensively with PBS. RNA was isolated from the cells,and the relative number of internalized WNV genome copies wasdetermined by qRT PCR. We observed an 85% reduction in the amountof internalized WNV RNA in MBCD-treated cells compared to thatin mock-treated samples (Fig. 4A). These results demonstratethe requirement for cholesterol-rich membrane microdomains inthe internalization of WNV. To further determine that the observedeffect was due to cholesterol depletion and not due to a pleiotropiceffect of MBCD on other membrane components, we supplementedcholesterol-depleted cells with exogenous cholesterol in thesame experiment. As shown in Fig. 4A, the addition of cholesterolto the cholesterol-depleted cells completely eliminated thedefects in WNV entry and restored normal viral titers in theculture supernatants (Fig. 4B). These data further indicatethat the plasma membrane cholesterol plays an important rolein WNV entry.

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FIG. 4. Cholesterol depletion affects WNV entry, and the effect can be eliminated by cholesterol substitution. (A) Vero cells were treated with MBCD as described above. For cholesterol (Chol) reconstitution, MBCD-treated cells were incubated in the presence of medium containing 400 µg of water-soluble cholesterol/ml in 2 mM MBCD for 1 h. After extensive washing, cells were infected at an MOI of 5 PFU/cell for 1 h at 37°C. RNA was isolated from cells, and WNV genome copy numbers were determined by qRT-PCR as described in Materials and Methods. Error bars represent SD (n = 10). (B) Vero cells were treated as described in the legend to panel A, and viral titers in the culture supernatants 24 hpi were determined by plaque assays. Error bars represent SD (n = 3). Results representative of at least two independent experiments performed with triplicates are shown.

WNV has been proposed to enter cells by endocytosis in clathrin-coatedvesicles. ${alpha}$ vβ3 integrin was identified previously as a receptorfor WNV, and it was shown that the internalization of WNV leadsto the activation of FAK (5, 6). We tested the requirement for ${alpha}$ vβ3 integrin and FAK in WNV infection by infecting wild-typeMEFs and MEFs lacking β3 integrin or FAK and found no differencein the viral titers in the culture supernatants of infectedcells between the control and knockout MEFs (Fig. 5A and B),suggesting that WNV is capable of infecting and replicatingin cells lacking ${alpha}$ vβ3 integrin and FAK. We further infectedhamster melanoma CS-1 cells that have been shown previouslyto lack ${alpha}$ vβ3 integrin and ${alpha}$ vβ5 integrin complexes atthe plasma membrane (11, 37) with WNV and measured viral titersin the supernatant 24 hpi. WNV efficiently infected CS-1 cells,and the titers in the supernatant were comparable to those inthe supernatants of the other cell lines used in this study(Fig. 5C). These results suggest that WNV can utilize an alternatereceptor in the absence of ${alpha}$ vβ3 or ${alpha}$ vβ5 integrin andthat these integrins are not essential for WNV infection.

WNV particles fail to be internalized in cholesterol-depleted cells. We next examined if the effect of MBCD on WNV entry is due tothe inefficient attachment of the virus at the cell surface.MBCD-treated cells were incubated with the virus at 4°Cfor 1 h to allow for binding, after which the unbound viruswas removed and the amount of bound virus was measured by determiningthe WNV genome copy numbers by qRT-PCR. As shown in Fig. 6A,cholesterol depletion did not affect the number of attachedvirions at the cell surface, suggesting that virus attachmentdoes not require lipid rafts. We further examined the endocytosisof WNV by infecting Vero cells at an MOI of 10 PFU/cell. Cellswere incubated with the virus at 4°C to allow virus attachmentand then shifted to 37°C for 30 min to allow internalization,after which the cells were subjected to fixation and immunofluorescencestaining with antibodies specific for WNV E and EEA1, a markerfor early endosomes. As shown in Fig. 6B, most of the internalizedWNV particles were in EEA1-positive early endosomes in controlcells, whereas no intracellular WNV particles could be detectedin MBCD-treated cells. A few of the WNV particles that werepresent in the cholesterol-depleted cells were at the cell peripheries.These data further confirm our qRT-PCR and viral titer dataand demonstrate that cholesterol depletion affects WNV internalization.

WNV E and CTB colocalize during entry. To investigate if WNV entry is mediated by cholesterol-richmembrane microdomains, we examined the internalization of WNVand FITC-conjugated CTB by Vero cells. The CTB entry pathwayhas been well-characterized, and CTB has been shown previouslyto enter cells in a lipid raft-dependent manner by binding toits receptor GM1 ganglioside (13). Vero cells were infectedwith WNV at an MOI of 10 PFU/cell and incubated with 10 µgof FITC-CTB/ml at 4°C for 30 min to allow for attachment.Cells were then shifted to 37°C for 10 min, washed extensively,fixed, and analyzed by immunofluorescence microscopy using anti-Eantibodies. We observed discrete areas of colocalization ofWNV E and CTB at the plasma membrane (Fig. 7). These data suggestthat both WNV E and CTB associate with lipid rafts at the plasmamembrane and that lipid rafts are required for the initial stagesof WNV internalization.

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FIG. 7. WNV E localizes with CTB at the plasma membrane. Vero cells grown on coverslips were infected at an MOI of 10 PFU of WNV/cell and incubated with 10 µg of FITC-conjugated CTB/ml for 30 min on ice. Cells were shifted to 37°C for 10 min, after which they were fixed in 4% PFA and stained with WNV E antibodies, followed by Alexa 594-conjugated secondary antibodies. The nuclei were stained by DAPI. The inset shows a magnification of the marked region of interest in the merged image. Scale bars, 10 µm.

DISCUSSION

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DISCUSSION

In this study, we showed that cholesterol-rich membrane microdomainsat the plasma membrane were important for normal WNV growth.The depletion of plasma membrane cholesterol from cultures ofvarious cell lines by MBCD affected WNV titers in the culturesupernatants. We observed a reduction in (i) the number of WNV-positivecells, as detected by immunofluorescence analysis, (ii) theamounts of viral proteins, and (iii) the levels of WNV RNA incholesterol-depleted cells. The observed effects were due toa block in WNV entry into the cells. WNV particles in cholesterol-depletedcells were undetectable in our internalization assays, furthersuggesting that WNV failed to bind to its receptor or an attachmentfactor in these cell samples. Additionally, WNV particles colocalizedwith CTB, which utilizes lipid rafts for internalization, inmicrodomain clusters at the plasma membrane. Together, our datasuggest that WNV utilizes lipid rafts for entry by binding eitherto a receptor present in the lipid rafts or to an accessoryprotein (an attachment factor or a signaling molecule) in thelipid rafts that promotes WNV binding to the receptor and WNVinternalization.

We also showed that WNV entry was ${alpha}$ vβ3 integrin independentand that FAK was nonessential for WNV growth. Previous studieshave used a lab strain of WNV (Sarafend) to study WNV endocytosisand have identified ${alpha}$ vβ3 integrin as a receptor for WNV.WNV Sarafend is a nonpathogenic strain whose source, year ofisolation, and passage history are not known. Phylogenetic analysesbased on sequences of E and NS5 proteins and 5' ends of the3' untranslated regions of multiple WNV isolates have been usedto classify WNV Sarafend as a lineage II WNV strain. WNV Sarafendalso falls into a separate antigenic group, as anti-E monoclonalantibodies recognizing all other WNV isolates fail to recognizeWNV Sarafend (32). It is possible that this virus differs fromthe infectious WNV strain NY99 that we used in our study inits receptor usage. This theory is further supported by thefact that while WNV Sarafend is reportedly unable to infectCS-1 cells that lack ${alpha}$ vβ3 integrin (5), the infection ofthese cells with WNV NY99 resulted in high viral titers comparableto those in Vero cells. While our studies were in progress,Mackenzie et al. reported that infection with Kunjin virus (anonpathogenic Australian variant of WNV) induces cholesterolbiosynthesis and that the viral replication is inhibited bydrugs that inhibit cholesterol biosynthesis (lovastatin) butnot by drugs that deplete cellular cholesterol (MBCD) (21).We have shown that, unlike Kunjin virus, WNV does not significantlyinduce cholesterol biosynthesis and that cholesterol depletionaffects WNV at the level of entry. In all the experiments describedby Mackenzie et al., MBCD or other drugs were added 90 min postadditionof virus and were left on the cells for the duration of theexperiment. Therefore, any effect of cholesterol on WNV entrywould not be observed under their experimental conditions. Theyfound that cholesterol depletion by MBCD postentry has no effecton Kunjin virus RNA replication or protein expression, but aroughly 10-fold reduction in viral titers was observed uponprolonged MBCD treatment, suggesting a possible role for cholesterolin later stages of the viral life cycle, such as assembly andegress. In our experiments, we added MBCD for 30 min at 3 or6 hpi and then removed the drug (we found prolonged exposureto result in significant toxicity), and we have not observedan effect on viral genome copies or titers. We cannot rule outthe possibility of the presence of residual intracellular cholesterolin the cells sufficient to participate in WNV replication. Alternatively,it is possible that cellular cholesterol levels were replenishedby de novo synthesis upon the removal of the drug, allowingfor cholesterol participation in viral replication, assembly,or egress.

Our results do not dispute the role of clathrin in WNV endocytosis(6, 19) but suggest that the lipid rafts are also required forWNV internalization. Several viruses from the family Flaviviridaehave been shown previously to exploit lipid rafts for entry(DENV, hepatitis C virus [HCV], and bovine viral diarrhea virus)or replication (Kunjin virus and HCV) (1, 17, 18, 21, 29). TheHCV receptors tetraspanin CD81 and scavenger receptor B typeI localize at specialized plasma membrane domains enriched withcholesterol. CD81-mediated HCV entry was shown previously torequire plasma membrane cholesterol (17). Similarly, Hsp90 andHsp70 have been proposed to form a receptor complex for DENV,and these proteins were shown previously to be associated withthe lipid rafts (29). In all these studies, the depletion ofplasma membrane cholesterol by MBCD led to a reduction in theviral titers in culture supernatants. Furthermore, membranefusion studies involving fusion between the liposomes and flavivirusor alphavirus virions have shown that cholesterol plays a crucialrole in promoting fusion, suggesting that the involvement ofcholesterol-rich membrane microdomains in viral entry may bea general feature of the entry pathways of flaviviruses andthe closely related alphaviruses (20, 34).

In addition to entry, viruses utilize lipid rafts at variousother stages of the life cycle. HCV and human immunodeficiencyvirus have been shown previously to modify the compositionsof the lipid rafts so as to facilitate viral replication (2,23). Lipid rafts act as signaling platforms due to the associationof signaling proteins, such as various growth factor receptors,Src family kinases, and GTPases, in these membrane microdomains(12). Therefore, hijacking lipid raft machinery by using oneof the lipid raft-associated proteins as a receptor or an attachmentfactor would enable the virus to gain access to various componentsof signaling and protein-trafficking machinery in the lipidrafts for later stages of viral replication (for an examplewith poliovirus, see reference 8). Further studies are requiredto examine how lipid rafts facilitate WNV growth and if WNV,like HCV, modifies host cell lipid raft compositions at latestages of virus replication. There is increasing evidence thata number of viruses utilize caveolin-independent, cholesterol-and clathrin-dependent endocytic pathways for internalization(14, 24, 38). Understanding the relative roles of clathrin-dependentand clathrin-independent endocytic processes in WNV internalizationwarrants further investigation. Identifying other proteins ofthe endocytic pathway that are involved in WNV internalizationwill provide further insights into the mechanism of WNV entryand the pathway involved. Our results demonstrate the importanceof plasma membrane cholesterol for WNV entry. This observationprovides new opportunities to further understand the mechanismsof WNV entry and replication and also to develop reagents thatcan block WNV entry and prevent WNV disease in the susceptiblehost.

ACKNOWLEDGMENTS

We thank Richard Hynes, David Cheresh, and David Schlaepferfor providing the β3 integrin^–/– MEFs, CS-1cells, and FAK^–/– MEFs, respectively. We thank membersof the Johnson lab for various reagents and members of the Nelsonlab for useful discussions. We thank Peter Mason for many usefulsuggestions and for critical reading of the manuscript.

This project has been funded in whole or in part with federalfunds from the National Institute of Allergy and InfectiousDiseases, National Institutes of Health, Department of Healthand Human Services, under contract no. HHSN266200500027C andby National Institutes of Health grant AI 61527 (to J.A.N.).

We declare no conflict of interest.

FOOTNOTES

* Corresponding author. Mailing address: Vaccine and Gene Therapy Institute, Oregon Health and Science University, 505 N.W. 185th Ave., Beaverton, OR 97006. Phone: (503) 494-2434. Fax: (503) 494-6862. E-mail: medigesh{at}ohsu.edu

Published ahead of print on 2 April 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

REFERENCES

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