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Journal of Virology, July 2008, p. 6470-6480, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00117-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cholesterol Effectively Blocks Entry of Flavivirus {triangledown}

Chyan-Jang Lee,1 Hui-Ru Lin,1 Ching-Len Liao,2 and Yi-Ling Lin1,2,3*

Institute of Biomedical Sciences,1 Genomics Research Center, Academia Sinica,3 Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China2

Received 17 January 2008/ Accepted 11 April 2008


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ABSTRACT
 
Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells through receptor-mediated endocytosis and low pH-triggered membrane fusion and then replicate in intracellular membrane structures. Lipid rafts, cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion with methyl-β-cyclodextrin or cholesterol chelation with filipin III reduces JEV and DEN-2 infection, mainly at the intracellular replication steps and, to a lesser extent, at viral entry. Using a membrane flotation assay, we found that several flaviviral nonstructural proteins are associated with detergent-resistant membrane structures, indicating that the replication complex of JEV and DEN-2 localizes to the membranes that possess the lipid raft property. Interestingly, we also found that addition of cholesterol readily blocks flaviviral infection, a result that contrasts with previous reports of other viruses, such as Sindbis virus, whose infectivity is enhanced by cholesterol. Cholesterol mainly affected the early step of the flavivirus life cycle, because the presence of cholesterol during viral adsorption greatly blocked JEV and DEN-2 infectivity. Flavirial entry, probably at fusion and RNA uncoating steps, was hindered by cholesterol. Our results thus suggest a stringent requirement for membrane components, especially with respect to the amount of cholesterol, in various steps of the flavivirus life cycle.


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INTRODUCTION
 
Flaviviruses are enveloped, single-stranded, positive-sense RNA viruses comprising many important human pathogens, such as yellow fever virus, West Nile virus (WNV), dengue virus (DEN), and Japanese encephalitis virus (JEV) (7). Flaviviral virions attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis (3, 37). The internalized virions then undergo conformational changes triggered by acidification of the endosomal vesicles, fusion of the viral and cell membranes, and particle disassembly (26, 37, 47). Translation of the released viral genome produces proteins required for RNA replication and for viral particle assembly (37, 38). Flaviviral infections induce dramatic intracellular membrane rearrangement and proliferation, during which viral RNA replication and virion maturation take place; thus, membranes are involved in every stage of the virus life cycle from the initial virus-cell encounter to the final release of viral particles.

The basic structure of the cell membrane includes a lipid bilayer comprised mainly of three different classes of lipids: phosphoglycerides, sphingolipids, and sterols. In the early fluid mosaic model, the cell membrane was viewed as a mosaic made of proteins inserted into the fluid lipid bilayer (68). In a later model, the membrane was thought to contain patches of lipid domains, whose composition and physical state differ from the average state of the bilayer (29). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids that accumulate in liquid-ordered, detergent-resistant membrane (DRM) domains (6, 67). Because of their ability to recruit or exclude specific lipids and proteins, lipid rafts have been implicated in the regulation of various physiological processes, such as lipid sorting, protein trafficking (45), cell polarization (28), and signal transduction (25, 51, 66).

Recent evidence suggests that the cholesterol-rich lipid rafts are involved in various steps of the life cycle of many enveloped and even nonenveloped viruses. During viral entry, lipid rafts may serve as the platform to concentrate virus receptors, to traffic the virus to the proper intracellular sites, and to affect the conformational changes in the envelope proteins during the fusion process (8, 42, 58). Lipid rafts also offer an efficient system for concentrating the virus proteins required for virion assembly, and many enveloped viruses bud from rafts (5, 8, 42). Moreover, it has been suggested that hepatitis C virus (HCV) RNA replication occurs on a lipid raft membrane structure, which requires the existence of cholesterol (1, 64).

During the flaviviral life cycle, cholesterol-rich membrane rafts have been shown to mediate DEN viral entry (61) and to trigger flavivirus-induced Akt phosphorylation (32). Recently, it has also been shown that WNV infection induces redistribution of cellular cholesterol and that changes in cholesterol biosynthesis and/or trafficking affect WNV RNA replication (41). The lipid requirements for flavivirus fusion have not been studied in the same detail as those for structurally similar alphaviruses. In the liposomal model system and in cholesterol-depleted cells, membrane fusion of Sindbis virus (SIN), a member of the alphavirus family, is absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane (39, 69). The involvement of lipids in flavivirus fusion has been studied using tick-borne encephalitis virus and a liposomal model system in which the presence of cholesterol in the target membrane, although not absolutely essential, was found to facilitate the membrane binding and trimerization of tick-borne encephalitis virus envelope protein (13, 70). However, the exact role of cholesterol in flaviviral fusion has not been revealed in a cell-based study.

In the present report, we define the roles of cholesterol in various steps of JEV and DEN serotype 2 (DEN-2) infections by use of cholesterol depletion as well as supplementation of extra amounts of cholesterol to cultured cells. We found that flaviviral entry, RNA uncoating, and replication could be blocked by either removal or addition of cholesterol, suggesting that fine-tuning of cholesterol levels in the target membranes is required for productive flaviviral infection. Our findings may lead to the possibility of a new potential antiviral strategy against the troublesome threat of the reemerging flaviviruses.


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MATERIALS AND METHODS
 
Cell lines, viruses, and chemicals. N18, a mouse neuroblastoma cell line (2), and baby hamster kidney BHK-21 were cultured in RPMI-1640 medium containing 5% fetal bovine serum (FBS) and 2 mM L-glutamine. JEV strain RP-9 (10), DEN-2 strain PL046 (36), and SIN-green fluorescent protein derived from the infectious clone dsTE-12Q (34) were used in this study. Virus propagation was carried out using C6/36 cells and RPMI-1640 medium containing 5% FBS. Methyl-β-cyclodextrin (MβCD) (C4555), filipin III (F4767), and a water-soluble cholesterol (C4951) that was balanced with MβCD at a ratio of ~1 mol of cholesterol to 5.68 mol of MβCD, which, according to a previous study (12) would result in cholesterol enrichment, were obtained from Sigma.

Virus infection and titration. Monolayers of cells in 6- or 12-well plates were adsorbed with virus at the indicated multiplicity of infection (MOI) for 1 h at 37°C. After adsorption, unbound virus was removed by gentle washing with serum-free medium followed by the addition of fresh medium and further incubation at 37°C. To determine virus titers, culture media were harvested for plaque-forming assays. Various virus dilutions were added onto 80% confluent BHK-21 cells and incubated at 37°C for 1 h. After adsorption, the cells were washed and overlaid with 1% agarose (SeaPlaque; FMC BioProducts) containing RPMI-1640 medium with 2% FBS. After incubations of 2 days for SIN, 4 days for JEV, and 7 days for DEN-2, cells were fixed with 10% formaldehyde and stained with 0.5% crystal violet.

Immunofluorescence assay (IFA). Cells were fixed with methanol/acetone (1:1) for 5 min at room temperature (RT) and then stained with primary antibodies at RT for 1 h. The antibodies used in this assay were anti-JEV NS1 and anti-DEN-2 NS1 antibodies (10, 36). After being washed with phosphate-buffered saline (PBS), the cells were reacted with secondary antibodies and examined with a Leica fluorescent microscope.

LDH assay. Cytotoxicity was assessed by the release of cytoplasmic enzyme lactate dehydrogenase (LDH) by use of a commercial kit (cytotoxicity detection kit; Roche). The culture supernatants from cell samples were clarified by centrifugation, mixed with a reaction mixture (diaphorase/NADH+ and tetrazolium salt INT [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride]/sodium lactate), incubated at RT for about 30 min, and then read by an enzyme-linked immunosorbent assay reader at 490 nm (Molecular Devices).

Intracellular staining and flow cytometric analysis. The cells were trypsinized, washed with PBS, and fixed with 4% formaldehyde for 20 min at RT. The cells were washed twice with ice-cold PBS-bovine serum albumin (BSA)-azide buffer (0.5% BSA and 0.1% sodium azide in PBS) and resuspended in permeabilization buffer (PBS-BSA-azide buffer containing 0.1% saponin) for 10 min at RT. The cells were resuspended in permeabilization buffer containing anti-NS3 antibodies (10, 36) for 30 min at RT. Alexa Fluor 488 goat anti-mouse immunoglobulin G (Molecular Probe) was then used as the secondary antibody to stain the cells for 30 min at RT in the dark. The cells were washed twice with permeabilization buffer and PBS-BSA-azide buffer and resuspended in PBS-BSA-azide buffer for flow cytometric analysis using a FACSCalibur system (BD Biosciences).

XTT assay. To determine the cell viability, a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based assay was performed (using cell proliferation kit II; Roche) according to the manufacturer's instructions. The cells were incubated with the reaction mixture at 37°C for about 30 min and then read by an enzyme-linked immunosorbent assay reader (Molecular Devices) at 490 nm.

Membrane flotation assay and Western blotting. The membrane flotation assay was performed by two different methods. The first was performed as reported previously (35). Briefly, cells were lysed in 2.5 ml of TNE buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, and 1 mM EDTA) containing 1% Triton X-100 at 4°C for 30 min and then passed through a 25-gauge needle 20 times. Nuclei and unbroken cells were removed by centrifugation at 700 x g for 5 min in a microcentrifuge at 4°C. Cell lysates were then mixed with 2.5 ml of 80% sucrose in TNE buffer and overlaid with 5 ml of 30% sucrose-TNE followed by 2.5 ml of 5% sucrose-TNE. The sucrose gradient was centrifuged at 40,000 rpm in a Beckman SW41 Ti rotor for 16 h at 4°C. After centrifugation, the floating bands of lipid raft were aspirated using a syringe and then dialyzed in 1 mM Tris-HCl (pH 7.4). A total of 1/20 of the sample was separated by 12% sodium dodecyle sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane (Hybond-C Super; Amersham Biosciences). In the second method (64), the cells were first lysed in 1 ml of hypotonic buffer (10 mM Tris-HCl [pH 7.5], 10 mM KCl, and 5 mM MgCl2) containing 1% Igepal CA-630 (Nonidet P-40) at 4°C for 1 h and then passed through a 25-gauge needle 20 times. Nuclei and unbroken cells were removed by centrifugation at 1,000 x g for 5 min in a microcentrifuge at 4°C. Cell lysates were then mixed with 3 ml of 72% sucrose in low-salt buffer (LSB; 50 mM Tris-HCl [pH 7.5], 25 mM KCl, and 5 mM MgCl2) and overlaid with 4 ml of 55% sucrose in LSB followed by 1.5 ml of 10% sucrose in LSB. The sucrose gradient was centrifuged at 38,000 rpm in a Beckman SW41 Ti rotor for 14 h at 4°C. After centrifugation, 1 ml fractions were taken from the top of the gradient, and each was added with 1.7 ml of LSB to dilute the sucrose and concentrated by passing through a Centricon YM-10 filter, resulting in about 100 µl. A tenth of each sucrose gradient fraction was separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with skim milk and incubated in primary antibodies against various JEV and DEN-2 proteins (10, 36). Anti-caveolin 2 antibody, which specifically recognizes amino acid residues 1 to 19 of the human and mouse caveolin 2 protein, was obtained from Calbiochem (catalog no. 219383). Finally, the blots were developed using an enhanced chemiluminescence system (Amersham Biosciences).

Isolation and amplification of viral RNA. N18 cells were adsorbed with DEN-2 in the presence or absence of cholesterol (100 µg/ml) for 1 h. To remove extracellular virions, cells were washed twice with serum-free RPMI medium and trypsinized for 5 min. The cells were then washed twice with culture medium and incubated at 37°C. The RNA was harvested at indicated time points using a viral RNA mini kit (ViroGen). Positive-sense viral RNA was reverse-transcribed using a ThermoScript reverse transcription kit (Invitrogen) with a primer annealing to DEN-2 nucleotides (nt) 10723 to 10703 (5'-AGAACCTGTTGATTCAACAGC-3'). The cDNA were subsequently PCR amplified by 5'-CCATGGGTAACGGAGTGGTCAGAC-3' (nt 8525 to 8548) and 5'-TGCATGGAACTACAAGTACGCG-3' (nt 9754 to 9733). Actin (5'-TCCTGTGGCATCCACGAAACT-3' and 5'-GAAGCATTTGCGGTGGACGA-3') was used as a control. The PCR products were analyzed by 1.2% agarose gel electrophoresis and visualized with ethidium bromide staining.

Establishment of JEV replicon cell line. JEV replicon construct was generated by cloning of the JEV RP-9 cDNA into pBR322 under the control of an SP6 promoter as outlined (see Fig. 9A). The internal ribosomal entry site (IRES)-neomycin resistance gene (Neo) cassette was inserted at the NsiI site in the 3' untranslated region (UTR) of JEV. BHK-21 cells were transfected with the in vitro-transcribed replicon RNA and selected with G418 (400 µg/ml). For colony formation, the replicon cells were cultured with G418 containing an agarose overlay plus different concentrations of cholesterol and were assessed by crystal violet staining after 1 week of incubation. The crystal violet-stained cells were lysed by 1% SDS and read by a spectrophotometer at 570 nm.


Figure 9
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  		FIG. 9. JEV replicon cell growth is slightly reduced by cholesterol treatment. (A) Schematic representation of the JEV replicon construct. SP6, SP6 promoter. "5' UTR" and "3' UTR" represent the 5' and 3' UTRs, respectively. C21 corresponds to the first 21 amino acids of JEV core protein, and E30 corresponds to the last 30 amino acids of JEV E protein. NS1-NS5 corresponds to the sequence coding for the JEV nonstructural proteins. IRES-Neo represents a sequence of an IRES of encephalomyocarditis virus followed by a neomycin resistance gene (Neo). (B) Establishment of a JEV replicon cell line. BHK-21 cells were transfected with in vitro-transcribed replicon RNA and selected with G418. The G418-resistant cells were stained with anti-JEV NS1 plus fluorescein isothiocyanate-conjugated secondary antibody and DAPI. (C) Colony formation of JEV replicon cells in the presence of cholesterol. The replicon cells and the pcDNA3-transfected BHK-21 cells were cultured with a G418-containing agarose overlay plus different concentrations of cholesterol as listed at the top of the panel. Colony formation was assessed by crystal violet staining after 1 week of incubation. (D) Replicon cell colony formation was slightly reduced by cholesterol treatment. The crystal violet-stained cells were lysed, and the level of optical density absorbance at an optical density of 570 nm was determined. The data are derived from the average results obtained with four plates for each experimental group. (E) JEV replicon cell growth was reduced by cholesterol treatment. The indicated cells were cultured with G418 plus cholesterol at different concentrations for 24 h before the cell growth was monitored by XTT assays. The averages and standard deviations of the results obtained with triplicate samples are shown.


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RESULTS
 
Depletion of cholesterol by MβCD affects JEV and DEN-2 infection. To study the role of cholesterol in flavivirus infection, we used MβCD to deplete cholesterol in the cells. MβCD is a derivative of cyclic oligosaccharides and has a lipophilic property that extracts cholesterol from membranes, resulting in lipid raft disruption (53). We first tested the drug effect of MβCD in mock-infected mouse neuroblastoma N18 cells. The cytotoxicity measured by LDH release and cell proliferation determined by XTT assays did not differ significantly between cells treated with MβCD at concentrations of up to 4 mM for 24 h and untreated cells (Fig. 1B). We then tested the potential antiviral effects of MβCD on flaviviral infection by treating the cells 1 h before viral adsorption and then throughout the infection period (–1 to 24 h). Viral protein expression detected by the anti-NS1 antibody (Fig. 1A) and infectious viral production determined by the plaque formation assay (Fig. 1C) decreased markedly in a dose-dependent manner in cells treated with MβCD. As SIN requires cholesterol for viral entry and exit (39), we included SIN as a control. SIN viral titers also decreased by up to 1.5 logs of magnitude in cells treated with 3 mM of MβCD (Fig. 1C). Because lipid raft may be involved in various steps of viral infection, we further defined the steps of flavivirus life cycle affected by MβCD by adding the compound to the cells before, during, or after viral entry. As shown in Fig. 1D and E, viral protein expression decreased the most when cholesterol was depleted after viral entry, suggesting that lipid rafts play a more important role in the steps after viral entry. The viral titers confirmed this result. Adding MβCD before or during viral entry decreased viral titer by about 10-fold, whereas adding MβCD after viral entry decreased viral titer by about 100-fold (Fig. 1F). Our results confirm that cholesterol depletion affects flaviviral entry, as previously reported (61); furthermore, they suggest that it plays an even more important role in the intracellular replication steps of JEV and DEN-2 infections.


Figure 1
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  		FIG. 1. Depletion of cholesterol by MβCD affects JEV, DEN-2, and SIN infection. (A and C) N18 cells were pretreated with MβCD (0, 1, 2, or 3 mM) for 1 h, and then the cells were adsorbed with JEV, DEN-2, or SIN (MOI = 10) in the presence of various concentrations of MβCD at 37°C. At 90 min after adsorption, the inoculated virus was washed away and the cells were cultured in fresh medium containing MβCD. At 24 h after infection, the cells were stained for IFA using anti-NS1 antibody to visualize viral protein expression (green) (A), and the culture supernatants were harvested for viral titration as determined by a plaque-forming assay (C). The cell nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole) (blue). (B) The drug effect of MβCD was determined in the mock-infected N18 cells by culturing with MβCD (0 to 5 mM) for 1 or 24 h, and cell proliferation and cytotoxicity were detected by XTT and LDH assays, respectively. O.D., optical density. (D) In the "Cell Pretreatment" group, N18 cells were pretreated with MβCD (5 mM) for 1 h, washed, and infected with JEV or DEN-2 (MOI = 10). In the "During Viral Entry" group, N18 cells were adsorbed with JEV or DEN-2 (MOI = 10) in the presence of MβCD (5 mM) at 37°C. After 90 min of viral adsorption, the cells were washed and then cultured in fresh medium without MβCD. The IFA and viral titration assay were performed at 24 h after infection. (E) In the "After Viral Entry" group, N18 cells were adsorbed with JEV or DEN-2 (MOI = 10) for 90 min at 37°C and then cultured in medium supplemented with MβCD (3 mM). The IFA and viral titration assay were performed at 24 h postinfection. (F) Severalfold decreases in titers of JEV and DEN-2 after treatment with MβCD. The viral production determined as shown in panels D and E is presented as severalfold reductions in viral levels compared with the results seen with cells not treated with MβCD. The data represent the averages and standard deviations of the results of two to three independent experiments.

Filipin III affects JEV and DEN-2 infections. To exclude the drug-specific effect of MβCD, we used another polyene macrolide antibiotic, filipin III, to examine the role of cholesterol in flaviviral infection. Cyclodextrins such as MβCD specifically disrupt rafts because of their ability to deplete cholesterol from membranes, whereas filipin III forms multimeric globular complexes with cholesterol within the cell membranes (24). Addition of filipin III to culture medium after viral entry decreased JEV and DEN-2 viral protein expression, especially when added at a concentration of 1 µg/ml (Fig. 2A). The viral production of JEV and DEN-2 was also decreased by up to 2 to 3 logs of magnitude by treatment with 1 µg/ml of filipin III (Fig. 2B). These results further highlight the role of lipid rafts and cholesterol during JEV and DEN-2 infection.


Figure 2
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  		FIG. 2. Filipin III blocks JEV and DEN-2 infection. (A and B) N18 cells were adsorbed with JEV or DEN-2 (MOI = 10) for 90 min at 37°C and then cultured in the medium supplemented with various doses of filipin III, as indicated. At 20 h postinfection, the cell lysates were immunoblotted with antibodies against NS1 or NS3 of JEV and DEN-2 or an actin control (A), and the culture supernatants were harvested and subjected to a plaque formation assay (B). (C) The drug effect of filipin in the mock-infected N18 cells was determined by culturing with filipin (0, 0.5, and 1 µg/ml) for 24 h, and then cell proliferation and cytotoxicity were detected by XTT and LDH assays, respectively. O.D., optical density.

Association of flaviviral nonstructural proteins with DRM fractions. Because lipid raft disruption by two different treatments blocked flaviviral replication at intracellular step(s) (Fig. 1 and 2), we speculated that JEV and DEN-2 replication takes place in membranes possessing lipid raft properties, as suggested previously for HCV (1, 16, 18, 56, 64). Several JEV and DEN-2 viral proteins such as NS1, NS3, and NS5, which are components of the replication complex (9, 14, 15, 40), were found to associate with the DRM fractions (Fig. 3A). A more detailed fractionation assay also showed that JEV NS1, NS3, and NS5 were present in the DRM fractions (Fig. 3B, lanes 6 to 9). As a control, caveolin 2, which is located in plasma membrane caveolae and is associated with lipid raft (17, 50, 54), was also present in the DRM fractions (Fig. 3B, lanes 7 and 8). These results indicate that several flaviviral nonstructural proteins are associated with the DRM structures that also contain caveolin 2 and have properties of lipid rafts.


Figure 3
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  		FIG. 3. Association of flaviviral nonstructural proteins with DRM fractions. (A) N18 cells were infected with JEV or DEN-2 (MOI = 10) for 18 h. The cells were lysed using 1% Triton X-100 in TNE buffer and then subjected to sucrose gradient ultracentrifugation. The floating bands were collected for Western blotting analysis as described in Materials and Methods. (B) N18 cells were infected with JEV (MOI = 10) for 16 h, and the cell lysates collected by use of 1% NP-40-LSB were subjected to sucrose gradient ultracentrifugation as described in Materials and Methods. The samples in each fraction were analyzed by Western blotting using the antibodies indicated on the right side of the panel.

The effects of MβCD are partially reversed by cotreatment with cholesterol. To confirm that MβCD acts through cholesterol depletion, we supplemented the MβCD-treated N18 cells with cholesterol. As expected, the viral entry of JEV and DEN-2 hindered by MβCD was reversed by cholesterol supplementation, as demonstrated by the results seen with both viral protein expression (Fig. 4, panels c) and infectious viral production (Fig. 4, panels d). However, the viral protein expression (Fig. 4, panels g), but not viral production (Fig. 4, panels h), blocked by MβCD after viral entry was rescued by addition of cholesterol. These results suggest that certain intracellular viral maturation step(s) may be affected by MβCD in a cholesterol-independent manner. Surprisingly, in the cholesterol control assay, we noticed a prominent antiviral effect in the cells treated with cholesterol during (Fig. 4, panels b and d) or after (Fig. 4, panels f and h) JEV and DEN-2 entry. The flaviviral protein expression measured by anti-NS1 antibody assays was almost completely eliminated by addition of cholesterol only during the 1.5 h of viral entry, leading to a drop in viral titer of up to 3 to 4 logs of magnitude.


Figure 4
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  		FIG. 4. The effects of MβCD are partially compensated by coincubation with cholesterol. In the "During Viral Entry" group, MβCD (5 mM) (a), cholesterol (50 µg/ml) (b), or MβCD (5 mM) plus cholesterol (50 µg/ml) (c) was added to the medium during JEV (A) or DEN-2 (B) (MOI = 5) adsorption at 37°C for 90 min. The cells were washed and cultured in fresh medium without any treatment. In the "After Viral Entry" group, after 90 min of JEV (A) or DEN-2 (B) (MOI = 5) adsorption at 37°C, the cells were cultured in medium with MβCD (5 mM) (e), cholesterol (50 µg/ml) (f), or MβCD (5 mM) plus cholesterol (50 µg/ml) (g). The viral protein IFA results (a, b, c, e, f, and g) and viral titer results (d and h) were determined at 24 h postinfection.

Plaque formation by JEV and DEN-2 in BHK-21 is affected by cholesterol. The findings presented in Fig. 4 were unexpected, because cholesterol is a cofactor for viral entry and enhances viral infection of murine hepatitis virus (72) and SIN (39). To further address the role of cholesterol in JEV and DEN-2 infections, we tested their plaque-forming abilities in BHK-21 cells in the presence or absence of cholesterol, with SIN used for control assays. As shown in Fig. 5, adding cholesterol during viral entry blocked plaque formation by JEV and DEN-2 but not that by SIN. Furthermore, the plaque sizes of JEV and DEN-2 were smaller when cholesterol was added after viral entry; this finding is in contrast to the results seen with SIN, whose plaque sizes were larger in the presence of cholesterol. These results indicate that cholesterol blocks the entry and replication steps of JEV and DEN-2 infection but enhances those of SIN.


Figure 5
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  		FIG. 5. Cholesterol decreases plaque formation of JEV and DEN-2 in BHK-21 cells. Viral plaque formation assays were performed using BHK-21 cells for JEV (A), DEN-2 (B), and SIN (C). The effect of cholesterol on viral entry was assessed by adding 50 µg/ml of cholesterol into the culture medium during viral adsorption at 37°C (middle panels). The cells were then washed and overlaid with agarose for plaque formation. The effect of cholesterol on viral replication was assessed by including cholesterol (50 µg/ml) in the agarose overlay (bottom panels). The plaques were stained with crystal violet as described in Materials and Methods.

Cholesterol blocks JEV and DEN-2 infection during virus entry and viral replication steps in N18 cells. We next determined the dosage effects and potential mechanism of cholesterol action in flaviviral infection. As N18 cells treated with cholesterol up to 100 µg/ml did not appear to exhibit a cytotoxic effect (Fig. 6A), the 50% cellular cytotoxicity of cholesterol on N18 cells is greater than 100 µg/ml. N18 cells infected with JEV or DEN-2 were treated with various doses of cholesterol at different times of infection. Pretreatment of cells with cholesterol had no noticeable antiviral effect (Fig. 6B and C, upper panels), indicating that cholesterol likely targets the virus particle but not the cell membrane. The viral protein expression and virus production of JEV and DEN-2 decreased markedly in a dose-dependent manner when cholesterol was added during or after viral entry (Fig. 6B and C, middle and bottom panels). It took about 11.8, 5.4, 2.7, and 3.2 µg/ml of cholesterol to reduce 50% of viral production for JEV and DEN-2 treated during and after viral entry; thus, the therapeutic index values for cholesterol treatment against JEV and DEN-2 could be greater than 8.5, 18.4, 37.6, and 31.8, respectively. These results suggest that cholesterol influences the entry and replication of JEV and DEN-2 in both N18 (Fig. 4 and 6) and BHK-21 (Fig. 5) cells.


Figure 6
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  		FIG. 6. Cholesterol blocks flaviviral infection at the viral entry and replication steps in N18 cells. (A) The drug effect of cholesterol was determined in the mock-infected N18 cells by culturing with cholesterol (0 to 100 µg/ml) for 1 or 24 h, and then cell proliferation and cytotoxicity were detected by XTT and LDH assays, respectively. O.D., optical density. (B and C) In the "Cell Pretreatment" group, N18 cells were pretreated with cholesterol (10 to 100 µg/ml) for 60 min, washed, and infected with JEV (B) or DEN-2 (C) (MOI = 10). In the "During Viral Entry" group, cholesterol (10 to 100 µg/ml) was added in the culture medium during viral adsorption at 37°C. In the "After Viral Entry" group, media with various doses of cholesterol (10 to 100 µg/ml) were used after viral entry. IFA with anti-NS1 antibody (red) and viral titration assays were performed at 18 h postinfection. The cell nuclei were stained with DAPI (blue).

Since the greatest antiviral effect of cholesterol was noted during the step of viral entry, we further studied how cholesterol might affect flaviviral infection. As shown in Fig. 7, cholesterol effectively blocked flaviviral infection regardless of whether it was added during viral adsorption at 4°C or even after viral binding at the entry step. To further examine whether the viral RNA uncoating is also affected by cholesterol, intracellular viral RNA was harvested and analyzed by reverse transcription-PCR. Treatment with cholesterol appeared to hinder the entry of DEN-2 viral RNA into the infected cells as early as 1 h postinfection and then appeared to hinder the subsequent viral RNA replication (Fig. 7D). Our results showing that cholesterol still blocked viral infection even after the viral adsorption step suggest that cholesterol might intercalate into the lipid bilayer of the virion envelope and lead to a hindrance of the downstream entry event. To support this notion, we pretreated the virus stock with cholesterol and then purified the viruses through a sucrose cushion ultracentrifugation procedure to remove the unattached cholesterol. The level of viral infectivity determined by plaque formation on BHK-21 cells indicated that ultracentrifugation through sucrose cushion did not hamper the antiviral potential of cholesterol (Fig. 8). These results suggest that cholesterol incorporation into virion affects the entry of flaviviruses, which hampers downstream viral RNA uncoating and viral replication during JEV and DEN-2 infection. To test whether cholesterol could suppress viral infection through a mechanism other than viral entry, we established a replicon cell line that contained a JEV replicon RNA carrying a neomycin-resistant gene as outlined in Fig. 9A. The replication of replicons renders the cells resistant to G418 selection, as reported for several other replicon systems. We found that even though the replicon cells can form G418-resistant colonies in the presence of cholesterol (Fig. 9C), the levels of colony formation (Fig. 9D) and cell growth (Fig. 9E) were less than those of control BHK-21 cells transfected with a pcDNA3 plasmid with a neomycin-resistant gene. This result is in accordance with our data shown in Fig. 6 indicating that cholesterol influences not only entry but also replication of flavivirus infection.


Figure 7
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  		FIG. 7. Cholesterol may block flaviviral entry and viral RNA uncoating. As outlined in panel A, BHK-21 cells were adsorbed with JEV (B) or DEN-2 (C) (MOI = 5) for 90 min on ice with or without cholesterol (100 µg/ml). The cells were washed twice with cold medium and incubated at 37°C in the presence or absence of cholesterol for 15 min. The cells were washed and incubated for 20 h before harvest for intracellular staining with anti-NS3 and flow cytometric analysis. (D) N18 cells were infected with DEN-2 (MOI = 5) for 60 min at 37°C with or without cholesterol treatment (100 µg/ml). The cells were then washed and incubated further for the times indicated (h p.i., hours postinfection). Total RNA was isolated and analyzed by reverse transcription-PCR for DEN-2 positive-sense viral RNA and actin as described in Materials and Methods.


Figure 8
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  		FIG. 8. Cholesterol may block flaviviral entry through intercalating into the lipid bilayer of virions. JEV and DEN-2 were incubated with cholesterol (0, 25, or 50 µg/ml) for 30 min and then ultracentrifuged through a 35% sucrose cushion at 35,000 rpm in a Beckman SW41 rotor for 3.5 h at 4°C. The pellets were then resuspended, and plaque levels were assayed using BHK-21 cells as described in Materials and Methods. The same batch of virus stock without ultracentrifugation was also treated with cholesterol (50 µg/ml) during the adsorption step of the plaque assay to serve as the positive control in the investigation of cholesterol's antiviral effect.


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DISCUSSION
 
In this study, we found that disruption of lipid raft formation by either MβCD or filipin III decreased JEV and DEN-2 infection, mainly at the intracellular replication step(s) (Fig. 1 and 2) and, to a lesser extent, at viral entry (Fig. 1). Consistent with this observation, we also found that several flaviviral nonstructural proteins such as NS1, NS3, and NS5 were associated with the DRM structures (Fig. 3), suggesting that flaviviral replication takes place in the lipid raft microdomains of the intracellular membrane. Interestingly, we also found that, in contrast to other viruses such as alphavirus and coronavirus, excess amounts of cholesterol appeared to block flaviviral infection (Fig. 4 to 6 and 8), probably because of a failure of viral entry and RNA uncoating (Fig. 7).

Cholesterol plays an important role in regulating cell membrane properties, and cellular cholesterol levels are tightly controlled by biosynthesis, efflux, and influx of cholesterol into cells (66). The distribution and dynamics of cholesterol in cells are also important in governing cholesterol's function. Lipid droplets, also called lipid bodies, were long thought of as inert cytoplasmic lipid inclusions but have been found recently to play a crucial role in maintaining the cellular cholesterol level by regulating lipid storage, hydrolysis, and trafficking (43, 44). Caveolins, the major components of cell surface caveolae (52), are thought to be involved in cellular cholesterol balance (27), and caveolin 1 has been shown to bind tightly with free cholesterol (48). Recently, caveolins were found on the surface of cytoplasmic lipid droplets (17, 50, 54), and the expression and trafficking of caveolins are regulated by cholesterol (27, 55). Thus, caveolin, cholesterol, and lipid droplets are functionally linked together in controlling cellular lipid balance. Interestingly, HCV capsid protein associates with lipid droplets in liver cells (46, 65), although the details of the involvement of intracellular lipid homeostasis in the life cycles of HCV and other viruses are largely unclear.

Studies have shown that extraction of cholesterol by MβCD inhibits the infectivity of many viruses. In human immunodeficiency virus infection, cholesterol depletion affects virus entry, thereby reducing the virus's infectivity (31, 57). Cholesterol is also thought to play a role in the conformational changes accompanying gp-41-mediated fusion (22). Entry of herpes simplex virus is inhibited by cholesterol-sequestering drugs such as MβCD and nystatin, which probably act by either preventing virus binding to the target cells or changing the receptor levels on the cell surfaces (4). Depletion of cholesterol by MβCD also inhibits rotavirus entry through a dynamin-dependent and caveolae-independent pathway (21, 63). Cholesterol requirement might well occur on the virus particle, since virion cholesterol depletion markedly reduces the fusion of influenza virus (71). As reported recently for DEN-2 (61), our results support the notion that lipid rafts on the plasma membrane facilitate entry of DEN-2; our results also apply to another flavivirus, JEV (Fig. 1 and 2). It is possible that depletion of cholesterol alters the integrity of raft microdomains, thereby affecting the binding or fusion efficiency, or both, of these flaviviruses.

In addition to virus binding and entry, cholesterol and lipid rafts seem to play a more important role in JEV and DEN-2 replication (Fig. 1 and 2). Accumulating evidence implies that flaviviral replication occurs in the intracellular membrane structures (73). In fact, genome replication of all positive-strand RNA viruses is believed to be associated with cellular membranes of the infected cells (62). The membrane association provides a structural framework for replication, with the advantages of increasing the local concentrations of necessary components and protecting the viral RNA molecules. Our results indicating that the viral nonstructural proteins (NS1, NS3, and NS5) are associated with the DRM (Fig. 3) suggest that the flaviviral replication complex may be located in the lipid raft structures of infected cell membranes. Recently, it has been shown for HCV, a member of the Flaviviridae family, that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes (1, 64). In hepatocyte cell lines containing an HCV RNA replicon, most of the nonstructural proteins, such as NS5A, NS5B, and NS3, were localized to the DRMs (18). Therefore, we suggest that as seen with HCV, the lipid raft membranes are also the sites of viral replication for JEV and DEN-2.

During entry of enveloped virus, cholesterol may play a role in promoting and stabilizing the local lipid bilayer bending that takes place during membrane fusion (11, 19, 23, 59, 69), and it is possible that cholesterol increases the infectivity of viruses through this mechanism. The plaque morphology of murine hepatitis virus is enlarged substantially after cholesterol supplementation (72). As is consistent with previous reports that SIN fusion is dependent on the presence of cholesterol in a liposomal model system and in cholesterol-depleted cells (39, 69), our results showed that the plaque-forming ability of SIN was enhanced by the cholesterol supplementation (Fig. 5C). However, the effect of this phenomenon was reversed for JEV and DEN-2, as cholesterol greatly blocked their ability to infect (Fig. 4 to 6). The fusion proteins of alphavirus and flavivirus, classified as class II viral fusion proteins (33, 60), are structurally similar, although their lipid requirements for fusion appear to differ (70). Alphavirus-mediated fusion is absolutely dependent on the presence of cholesterol and sphingolipids in the target membrane (30), whereas sphingolipids are not necessary at all for flavivirus fusion (13, 20). Cholesterol is also not absolutely required, although its presence increases the overall efficiency of the flaviviral fusion reaction in the liposome model system (70). Our experiment using the cell-based assay demonstrated a different role of cholesterol in members of the class II viral fusion proteins, as flaviviral entry and release of viral RNA were blocked by cholesterol supplementation (Fig. 7). We suspect that the presence of extra cholesterol in the virion membrane increases its rigidity (49, 74), resulting in a higher energy barrier for membrane fusion and leading to a freezing in the step of virus entry. In addition, cholesterol addition after viral entry still reduced JEV and DEN-2 replication (Fig. 6 and 9), suggesting that an intracellular step of flavivirus infection is also sensitive to the disturbance in cholesterol levels. Understanding in greater detail these blocking events caused by the presence of cholesterol would certainly provide more information about the life cycle of flaviviruses and might lead to development of new drugs against these viruses, which threaten millions in the world population annually.


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ACKNOWLEDGMENTS
 
Y.-L.L. is supported by grants from the National Science Council (NSC-95-2320-B-001-031-MY3 and NSC-96-3112-B-001-021) and from Academia Sinica, Taiwan, Republic of China.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biomedical Sciences, Academia Sinica, No. 128, Sec. 2, Academy Rd., Nankang, Taipei 11529, Taiwan, Republic of China. Phone: (886) 2-2652-3902. Fax: (886) 2-2785-8847. E-mail: yll{at}ibms.sinica.edu.tw Back

{triangledown} Published ahead of print on 30 April 2008. Back


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Journal of Virology, July 2008, p. 6470-6480, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00117-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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