Cholesterol-dependent infection of Burkitt's lymphoma cell lines by Epstein–Barr virus

Rebecca B. Katzman and Richard Longnecker

Department of Microbiology-Immunology, Northwestern University Medical School Chicago, IL 60611, USA

Correspondence
Richard Longnecker
r-longnecker{at}northwestern.edu


   ABSTRACT
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Epstein–Barr virus (EBV) infection is a multi-step process, first requiring virus binding to the host cell, followed by fusion of the viral envelope with the host cell plasma membrane. Efficient EBV entry into B cells requires, at the minimum, the interaction of the EBV-encoded glycoproteins gp350 with cellular CD21 and gp42 with MHC class II proteins. In this study, use of the cholesterol-binding drugs methyl-{beta}-cyclodextrin and nystatin efficiently inhibited EBV infection of target Burkitt's lymphoma B-cell lines, indicating an important role for cholesterol and suggesting the involvement of lipid rafts in EBV infection.


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Epstein–Barr virus (EBV) is a gammaherpesvirus whose only natural host is in humans and which is associated with many diseases (Kieff & Rickinson, 2001; Liebowitz, 1998; Longnecker, 1998; Rickinson & Kieff, 2001). EBV is associated with the haematopoietic cancers African Burkitt's lymphoma, Hodgkin's lymphoma and certain T-cell lymphomas, and with the epithelial diseases nasopharyngeal carcinoma, oral hairy leukoplakia and gastric carcinoma. Most humans are asymptomatically infected with EBV during childhood; however, EBV infections during adolescence can result in acute infectious mononucleosis. Following lytic infection and immune clearance of the majority of EBV-infected cells, the virus persists latently in B lymphocytes.

In vitro, EBV readily infects B cells by a receptor-mediated process (Nemerow & Cooper, 1984; Speck et al., 2000). Binding of gp350/220, the major EBV outer envelope glycoprotein, to the cellular complement receptor CD21 mediates initial attachment of the virus to B cells (Tanner et al., 1987). Following attachment, viral gp42 interacts with the EBV coreceptor cellular MHC class II proteins for viral fusion and penetration of cells (Haan et al., 2000; Spriggs et al., 1996; Wang & Hutt-Fletcher, 1998). gp42 also requires the concerted efforts of the additional viral glycoproteins gH, gL and gB for fusion of the two membranes (Haan et al., 2001; Haddad & Hutt-Fletcher, 1989; Li et al., 1995).

Both receptors for EBV, CD21 and MHC class II, utilize lipid rafts for cellular function. Lipid rafts are microdomains within the plasma membrane that have a distinct lipid and protein content, as well as high concentrations of cholesterol (Brown & Jacobson, 2001; Kurzchalia & Parton, 1999; Simons & Ikonen, 1997; Simons & Toomre, 2000). Lipid rafts have been shown to be sites for many viruses to infect their host cells [for example simian virus 40 (SV40), human immunodeficiency virus (HIV) and filoviruses] (Bavari et al., 2002; van der Goot & Harder, 2001; Mañes et al., 2003). Therefore, we hypothesized that EBV may require cholesterol-enriched domains in plasma membranes to infect B lymphocytes.

If EBV entry is dependent upon membrane cholesterol levels, then cholesterol depletion of cells should abrogate infection. Daudi B cells are an EBV-positive Burkitt's lymphoma cell line that is readily infected by EBV. We used these cells to assay for cholesterol dependence for EBV entry into B cells. First, we depleted Daudi B cells of membrane cholesterol with methyl-{beta}-cyclodextrin (MCD). MCD specifically sequesters cholesterol from the plasma membrane of cells, thereby disrupting lipid rafts (Ohtani et al., 1989; Scheiffele et al., 1997). Cells were pretreated for 15 min with increasing concentrations of MCD (0–10 mM). Cells were then infected with recombinant EBV (EBfaV–GFP) in the presence of MCD at 37 °C while rocking for 30 min. All infections were carried out in serum-free Opti-MEM medium. EBfaV–GFP has an EGFP cassette inserted into the viral genome (Speck & Longnecker, 1999). GFP is expressed only in cells that become infected by this virus and the rate of viral infection can be quantified using flow cytometry to detect GFP-positive cells 2 days post-infection. Upon treating cells with increasing concentrations of MCD, cholesterol depletion inhibited B cell infection in a concentration dependent manner (Fig. 1A, C).



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Fig. 1. Cholesterol depletion inhibits EBV infection of Daudi B cells. (A) Daudi B cells were pretreated with MCD at the indicated concentrations for 15 min and then infected with recombinant EBV (EBfaV–GFP) for 30 min while maintaining MCD concentrations. FACS analysis was performed 48 h after infection and GFP fluoresence was used to determine the percentage inhibition by MCD of EBV infection into Daudi cells. (B) Daudi B Cells were pretreated with MCD for 15 min, washed and then infected with EBfaV–GFP for 30 min without the presence of MCD. (C) Curves representing three independent experiments each showing the percentage inhibition by MCD of EBfaV–GFP into cholesterol-depleted cells (15 min pretreated) either in the presence of MCD ({blacktriangleup}) or without MCD (washed out as in B) ({bullet}). NA, Not applicable.

 
Next, we wanted to establish whether loss of EBV infection was due to cholesterol extraction from the cellular plasma membrane or from the viral envelope. Therefore, we repeated the above experiments; however this time the cells were washed free of MCD following pretreatment, and then infected as before. Again we saw significant inhibition of EBV infection of the cells; however the effect was not as great in this case when the MCD was washed out, as when the cyclodextrin was maintained during infection (Fig. 1B, C). These data demonstrate that Daudi B cells require membrane cholesterol in order to be infected by EBV.

Since MCD treatment of Daudi cells abrogated EBV entry, we wanted to confirm the availability of the EBV cellular receptors for virion binding. We tested whether MCD altered the levels of cell surface expression of the cellular receptors for EBV entry. HLA-DQ is the MHC class II protein we first checked. Cells were treated with increasing concentrations of MCD for 15 or 45 min, representing the times in which cells were exposed to MCD in Fig. 1. Treated cells were then stained with antibody recognizing HLA-DQ followed by a fluorochrome-conjugated secondary antibody. Antibody binding to cells was then detected using flow cytometry. Under all conditions there was no significant change in receptor cell surface expression induced by MCD treatment (Fig. 2A, B). The same result was also demonstrated using a pan-MHC class II primary antibody that detects HLA-DR, -DP, and -DQ (data not shown). Using a fluorochrome-conjugated primary antibody recognizing CD21, we showed that MCD did not downregulate the surface expression of CD21 either (Fig. 2B).



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Fig. 2. MCD treatment is not toxic for Daudi B cells: surface expression of EBV cellular receptors is unaffected and recovery of membrane cholesterol restores EBV infection. (A, B) Daudi B cells were either treated with MCD for 15 or 45 min. The cells were then stained with antibodies specific for HLA-DQ or CD21 followed by the appropriate secondary antibody and then analysed by flow cytometry. (A) Histogram plots representing the intensity of staining for HLA-DQ after MCD treatment for 15 or 45 min at increasing concentrations from one experiment. (B) Summary of three independent experiments monitoring HLA-DQ and CD21 expression. Change in the number of positive cells was determined by observing the percentage of cells within a specific gate with and without MCD treatment for the indicated times. The gate was selected based on HLA-DQ or CD21 expression of untreated cells. (C) Daudi cells were pretreated with the indicated concentrations of MCD for 15 min ({blacktriangleup}). For cholesterol recovery, cells were incubated for 4 h in serum-containing medium supplemented with water-soluble cholesterol ({bullet}). Cells were infected with EBfaV–GFP without the presence of MCD. FACS analysis was used to determine the number of cells expressing GFP and percentage inhibition of infection was determined. The graph represents data from three independent experiments.

 
We next wanted to assay for any effects of MCD on the cellular integrity of the treated cells. Viable cells are able to recover plasma membrane cholesterol levels following MCD treatment. Therefore, cholesterol-restored cells should be efficiently infected by EBV. Thus, we depleted plasma membrane cholesterol with increasing concentrations of MCD for 15 min, washed the cells and then allowed half the cells to recover in complete medium supplemented with MCD-conjugated cholesterol (MCD-chol) for 4 h. MCD forms water-soluble inclusion complexes with cholesterol (Klein et al., 1995). MCD-chol delivers cholesterol to cell plasma membranes. Both MCD-recovered and -unrecovered cells were infected with EBfaV–GFP as before, and then assayed for GFP expression by flow cytometry. At both 2·5 and 10 mM MCD, infection by EBV of Daudi cells was inhibited as in Fig. 1(B, C). However, cholesterol-recovered Daudi cells were efficiently infected by EBV (Fig. 2C). As a control for MCD treatment, untreated cells were also incubated with MCD-chol-supplemented complete medium for 4 h. EBV efficiently infected MCD-chol-treated cells (Fig. 2C). This confirms that MCD treatment does not inhibit infection by causing cell death.

Next we wanted to determine if the inhibition of infection induced by MCD treatment was specific for EBV entry, and not due to MCD cytotoxicity or unique to the Daudi cell line. Therefore Raji cells, another EBV-positive B cell line, were used in the next experiment. Raji cells were treated with increasing concentrations of MCD for 15 min, washed and then infected with EBfaV–GFP without the presence of MCD. Inhibition of EBV entry into these B lymphocytes was similar to that seen with Daudi cells (Fig. 3A). The rate of infection by EBV into MCD-treated Raji cells was also compared to the efficiency of infection of vesicular stomatitis virus (VSV) G protein-pseudotyped retrovirus infection. A non-replication competent retroviral vector that expresses GFP was used. VSV G protein-pseudotyping of retroviruses results in pantropic retroviruses (Yee et al., 1994). VSV G protein interacts with phospholipid components of the plasma membrane (Mastromarino et al., 1987). As such, receptor-mediated endocytosis by VSV G protein-pseudotyped retroviruses is lipid raft independent (Guyader et al., 2002; Popik et al., 2002). In three independent experiments, there was no defect in VSV G protein-pseudotyped retroviral entry of Raji cells following MCD treatment (Fig. 3A). These data show that MCD-treated B cells are still susceptible to virus infection; EBV, in contrast, requires membrane cholesterol to infect B cells.



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Fig. 3. EBV entry into B cells is specifically inhibited by cholesterol depletion. (A) Raji cells were pretreated with the indicated concentrations of MCD for 15 min. Half of the cells were then infected with EBfaV–GFP ({blacktriangleup}) and the rest were infected with VSV G protein-pseudotyped retrovirus expressing GFP ({bullet}). MCD was washed out prior to infection. FACS analysis was used to determine the number of cells expressing GFP and percentage inhibition of infection was determined. The graph represents data from three independent experiments. (B) Raji cells were pretreated with increasing concentrations of nystatin for 20 min. The cells were then infected and analysed as above. The graph represents the average of two independent experiments.

 
Nystatin is an antifungal reagent that also disrupts cholesterol-enriched microdomains; it does so by binding to cholesterol specifically (Bolard, 1986; Rothberg et al., 1990). Raji cells were pretreated with increasing concentrations of nystatin for 20 min at 37 °C while rocking. The cells were then infected with either EBfaV–GFP or VSV G protein-pseudotyped retrovirus as before. Similar to MCD treated cells, nystatin only inhibited entry of EBV particles into Raji cells, whereas VSV G protein-pseudotyped retrovirus entered the cells efficiently under all conditions (Fig. 3B). At the highest concentrations of nystatin tested, Daudi cells were also inhibited for entry of EBV by approximately 25 % (data not shown).

The current study using MCD and nystatin, both of which specifically target cholesterol in biological membranes, indicates that EBV requires cholesterol for efficient infection. Furthermore, since cholesterol is an important component of lipid rafts, our data suggest a role of lipid rafts in EBV entry. Previous studies have demonstrated that cholesterol and lipid rafts are essential for efficient entry of several viruses: influenza virus, filoviruses, HIV and SV40 (Bavari et al., 2002; van der Goot & Harder, 2001; Mañes et al., 2003). This is the first study implicating the involvement of cholesterol-enriched domains in EBV entry. Conversely, some endocytosed viruses do not require cholesterol for virus entry as demonstrated in this and previous studies of VSV G protein-pseudotyped retroviruses (Guyader et al., 2002; Popik et al., 2002). This has also been shown for alphavirus infection (Waarts et al., 2002). In the case of EBV entry, cholesterol may be important for membrane fusion, receptor localization in membrane microdomains and/or early viral signalling events. Although we cannot specifically exclude a post-entry event including uncoating, transport of the nucleocapsid to the nucleus, release of the viral genome into the nucleus, and subsequent viral gene expression, it would seem unlikely based on our results with the VSV G protein-pseudotyped retrovirus. In order for the GFP within the retroviral genome to be expressed, the virus capsid must transport to the nucleus where gene expression can initiate similar to the requirements for GFP expression from the recombinant EBV used in our studies.

Fusion of the EBV envelope with the cellular membrane may require a cholesterol-rich environment. Lipid rafts are microdomains in membranes that are highly enriched in cholesterol (Brown & Jacobson, 2001; Kurzchalia & Parton, 1999; Simons & Ikonen, 1997; Simons & Toomre, 2000). As a consequence, they are more rigid and highly ordered than the majority of cellular membranes. As such, they may provide an environment for enveloped herpesviruses, like EBV, to fuse with cells and, therefore, disrupting lipid rafts with MCD or nystatin abrogates viral infection.

MCD treatment of B cells did not affect the levels of cell surface expression of either CD21 or MHC class II; therefore the observed inhibition of infection is likely due to some other alteration induced by MCD. Lipid rafts have been shown to play a role in MHC class II protein function (Anderson et al., 2000; Bouillon et al., 2003; Hiltbold et al., 2003; Huby et al., 1999). EBV requires the binding of gp42 to MHC class II proteins to fuse its membrane with that of the host B-cell membrane. There is some controversy as to whether or not MHC class II proteins constitutively reside in lipid rafts or if they are recruited into the microdomains following activation. Despite this controversy, it appears that MHC class II proteins utilize lipid rafts for antigen presentation, signalling through the Src family protein tyrosine kinase Lyn and for accumulation at the immunological synapse (Anderson et al., 2000; Bouillon et al., 2003; Hiltbold et al., 2003; Huby et al., 1999). Similarly CD21, which EBV gp350 uses to bind to B lymphocytes, also employs lipid rafts for its function in B-cell signalling (Cherukuri et al., 2001).

Furthermore, in regard to signalling, lipid rafts function as specific sorting sites required for certain signal transduction pathways to occur. In many cases, it has been shown that upon ligand binding, receptors are recruited to lipid rafts and this recruitment is essential for proper signal transduction to emanate from the ligated receptors. EBV is thought to infect resting B cells trafficking through the oral mucosa and previous studies have demonstrated that the early stages of EBV infection activate cellular signal transduction (Fuller & Perez-Romero, 2002; Nemerow et al., 1994). Appropriately it has been shown that purified gp350 binding to CD21 results in the upregulation of TNF-{alpha} expression via protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and tyrosine kinase pathways signalling to the transcription factor NF{kappa}B (D'Addario et al., 2000). Thus, cholesterol depletion and the resulting effects on lipid rafts could prevent a specific, and necessary, early signalling event required for productive EBV infection. MCD and nystatin treatment of cells did not wholly inhibit virus entry but was specific to EBV; efficient infection by VSV G protein-pseudotyped retroviruses was unaffected indicating that cholesterol depletion did not adversely affect retroviral capsid transport to the nucleus and subsequent viral gene expression. VSV G protein-induced endocytosis is a pH-dependent process and not a signal transduction-dependent process (Hernandez et al., 1996). Further studies will be required to more fully address the role of cholesterol in EBV infection.


   ACKNOWLEDGEMENTS
 
We would like to thank Marisa McShane, Michelle Swanson-Mungerson and members of the Longnecker Lab for their help in performing these studies. R. K. was supported in part by a training grant from the National Institutes of Health (T32CA009560). R. L. is a Stohlman Scholar of the Leukaemia and Lymphoma Society of America and supported by Public Health Service grants CA62234, and CA73507, and CA93444 from the National Cancer Institute and DE13127 from the National Institute of Dental and Craniofacial Research.


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Received 24 March 2003; accepted 30 July 2003.



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