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Liposome-mediated transient transfection reduces cholesterol-dependent coxsackievirus infectivity
Journal of Virological Methods 133 (2006) 211–218
Liposome-mediated transient transfection reduces cholesterol-dependent coxsackievirus infectivity Jerry Wong, Jingchun Zhang, Guang Gao, Mitra Esfandiarei, Xiaoning Si, Yahong Wang, Bobby Yanagawa, Agripina Suarez, Bruce McManus, Honglin Luo ∗ Department of Pathology and Laboratory Medicine, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul’s Hospital, Vancouver, BC, Canada Received 11 September 2005; received in revised form 11 November 2005; accepted 15 November 2005 Available online 20 December 2005
Abstract Liposome-mediated gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Coxsackievirus B3 is an important human pathogen associated with various diseases. Here we reported that liposome-mediated transient transfection of plasmid cDNA inhibited coxsackieviral replication at the levels of RNA, protein and viral progeny release. These inhibitory effects were observed in various cell types and by using different liposome reagents. We further showed that the inhibition was likely due to the lack of virus attachment. Moreover, we showed that addition of cholesterol restored, at least in part, the viral infectivity. Interestingly, we found that membrane cholesterol levels were unchanged during transfection, indicating that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Our data suggest that liposome-mediated cDNA transient transfection inhibits coxsackievirus infectivity via inhibition of viral attachment, which is likely occurring through the changes of membrane cholesterol integrity. © 2005 Elsevier B.V. All rights reserved. Keywords: Liposome; Transient transfection; Coxsackievirus; Cholesterol; Lipid raft; Viral binding
1. Introduction Coxsackievirus B3 (CVB3) is a small, non-enveloped, positive-stranded virus belonging to the Enterovirus genus in the family of Picornaviridae. CVB3 is a common human pathogen associated with a broad spectrum of clinically relevant diseases including acute and chronic myocarditis, meningitis, pancreatitis and possibly insulin-dependent diabetes (Clements et al., 1995; McManus et al., 1991; Woodruff, 1980). Despite extensive efforts, knowledge regarding the mechanisms involved in coxsackievirus B3 infectivity, in particular the initial phase of viral–host interaction including viral attachment and internalization, is limited. The coxsackie-adenovirus receptor (CAR) (Bergelson et al., 1997; Martino et al., 2000) and the decay accelerating factor ∗
Corresponding author. The James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul’s Hospital, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6. Tel.: +1 604 682 2344x62847; fax: +1 604 806 8351. E-mail address: [email protected] (H. Luo). 0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.11.013
(DAF/CD55) (Martino et al., 1998; Shafren et al., 1995) have been identified as receptors for CVB3, which together facilitate cell binding and internalization of CVB3 into permissive host cells. CAR is a member of the junction adhesion molecule subgroup of the immunoglobulin superfamily (Cohen et al., 2001). Expression of CAR is highly regulated and generally correlated with viral tropism. DAF is a glycosylphosphatidylinositol (GPI)anchored protein, serving as a coreceptor for CVB3 infection. Recent studies have indicated that both CAR and DAF are localized to lipid-rich microdomains or lipid rafts of cell membranes, implicating that lipid rafts may be important for CVB3 attachment and entry (Ashbourne Excoffon et al., 2003; Stuart et al., 2002). Involvement of lipid rafts in viral replication has been reported in both enveloped viruses (human immunodeficiency virus (Campbell et al., 2001), influenza virus (Barman et al., 2004), Ebola virus (Bavari et al., 2002)), and non-enveloped viruses (simian virus 40 (Parton and Richards, 2003), echovirus 11 (Stuart et al., 2002), rotavirus (Cuadras and Greenberg, 2003), CVB4 (Triantafilou and Triantafilou, 2004), and coxsackievirus A9 (Triantafilou and Triantafilou, 2003)). Cholesterol is a critical component in lipid rafts which are involved in many cellular
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processes such as endo/exocytosis, intracellular trafficking, and membrane signaling (Alonso and Millan, 2001; Simons and Ehehalt, 2002). Gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Thus far, many techniques have been developed to deliver genes of interest into mammalian cells, which include viral gene transfer, electroporation, calcium phosphate co-precipitation, cationic lipids/DNA complexes, diethylaminoethyl-dextran/DNA, and polycation/dimethyl sulfoxide. Among them, cationic liposome-based transfection has been widely used for in vitro and in vivo gene delivery in eukaryotic cells due to the non-immunogenic and non-oncogenic properties, low cytotoxicity, and relatively high transfection efficiency (Zabner, 1997). The cellular and molecular mechanisms of cationic lipid-mediated gene transfer remains unclear. Early data suggested that cationic lipids-DNA complex (lipoplex) entered the cells by fusing with the cellular membrane (Noguchi et al., 1998). However, recent studies suggest that cationic liposome-mediated transfection of mammalian cells involves in an endocytotic process, occurring through a cholesteroldependent clathrin-mediated pathway (Zabner, 1997; Zuhorn et al., 2002). In a preliminary study, we observed a novel phenomenon that lipofectamine-mediated delivery of DNA inhibits CVB3 infectivity, which is independent of the properties of the vectors and genes. In this study, we further explore the viral inhibitory effect of cationic liposome-mediated transient transfection and attempt to define the mechanisms involved. We showed that CVB3 infectivity was reduced in HeLa cells which were transiently transfected by liposome. We further demonstrated that liposome–DNA complex suppression of viral infectivity was due, at least in part, to changes in the cellular cholesterol environment. 2. Materials and methods
of cells the next day using LipofectamineTM (Life Technologies) according to the manufacturer’s instructions. Briefly, lipofectamine (6 l) and DNA (1.5 g) were diluted in 100 l of Opti-MEM followed by equilibration at room temperature for 5–10 min after mixing. The lipofectamine–DNA complex was added to HeLa cells and incubated for 6 h. Cells were then washed with PBS and replenished with DMEM containing 10% serum. To examine transfection efficiency, cells transfected with a pcDNA-LacZ plasmid were lysed to determine the -galactosidase activity using a Promega kit according to the manufacturer’s recommendation. 2.3. Virus infection CVB3 (Nancy strain), a gift from Dr. Reinhard Kandolf (University Hospital of Tubingen, Tubingen, Germany), was propagated in HeLa cells and stored at −80 ◦ C. Virus titres were routinely determined by plaque assay on HeLa cell monolayers at the beginning of the experiments as described below. Cells were infected either with CVB3 at a multiplicity of infection (MOI) of 10 or with PBS (Sham). Following 1 h of incubation at 37 ◦ C, cells were washed with PBS and replenished with DMEM containing 10% serum. 2.4. Plaque assay The amount of CVB3 produced was measured on monolayers of HeLa cells by agar overlay plaque assay of supernatant cultures as previously described (Luo et al., 2002). Briefly, cell supernatant was serially diluted and overlaid on monolayers of HeLa cells. Following 1 h of incubation, medium was removed and complete DMEM containing 0.75% agar was overlaid. Three days post-infection, cells were fixed with Carnoy’s fixative (25% acetic acid, 75% ethanol) and then stained with 1% crystal violet. Viral titres were determined as plaque-forming units (PFU) per milliliter.
2.1. Cell culture and materials 2.5. Western blot analysis HeLa cells (HeLa S3) were obtained from the American Type Culture Collection. Subconfluent cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated newborn calf serum (Life Technologies) at 37 ◦ C in a humidified incubator with 5% CO2. Penicillin G (100 g/ml) and streptomycin (100 g/ml) (Life Technologies) were added to all culture media. Most supplies were purchased from Sigma Chemical Co. unless otherwise specified. The monoclonal antibody against CVB3 capsid protein 1 (VP1) was obtained from DakoCytomation. Monoclonal B-actin antibody, horseradish peroxidaseconjugated anti-rabbit and anti-mouse immunoglobulin G were purchased from Santa Cruz Biotechnology. The monoclonal anti-CAR antibody was from Upstate.
Western blot was performed as previously described (Luo et al., 2003). Briefly, equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech). The membrane was blocked with 5% non-fat dry milk solution containing 0.1% Tween-20 for 1 h. Afterwards, the membrane was incubated with primary antibody for 1 h, followed by secondary antibody for 45 min at room temperature. The immunoblots were visualized using an enhanced chemiluminescence detection system according to the manufacturer’s protocol (Amersham Pharmacia Biotech). 2.6. In situ hybridization
2.2. Transient transfection HeLa cells were plated onto six-well plates overnight and transfections were carried out on cells at 70–80% confluence
In situ hybridization was performed as previously described (Esfandiarei et al., 2004). Briefly, fixed cells were hybridized with digoxignenin-labeled CVB3 antisense riboprobes, which
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were prepared from the full-length CVB3 cDNA using an in vitro transcription kit (Promega) according to the manufacturer’s instructions. Hybridized riboprobes were detected using an alkaline phosphatase-conjugated anti-digoxignenin antibody (Roche Diagnostics) and a color substrate Vector Red (Vector Laboratories). Cells were counter stained with hematoxylin. 2.7. Virus radiolabeling and purification Confluent HeLa cells were infected with CVB3 at a MOI of 10 in methionine-free medium. Three hours after infection, medium containing 19.4 MBq/ml (525 Ci/ml) of tran [35 S] label (MP Biomedicals) was added for an additional 8 h. HeLa cells and medium were harvested and then frozen and thawed three times. The cellular debris was spun down and the supernatant was collected and centrifuged at 45,000 rpm for 90 min. The pellet containing [35 S]-labeled CVB3 was resuspended in serum-free DMEM and virus titer was measured by plaque assay and aliquots were stored at −80 ◦ C. 2.8. Virus binding assay Confluent HeLa cells were incubated with [35 S]-labeled CVB3 at a MOI of 1 (0.37 cpm/pfu) at 4 ◦ C for 1 h. The cells were washed thoroughly with PBS and lysed with 2% SDS. The amount of [35 S]-labeled CVB3 in the samples was measured with a 1217 rackbeta liquid scintillation counter (LKB Wallac). 2.9. Immunocytochemical staining At 48 h after transient transfection, HeLa cells were fixed with 4% paraformaldehyde, permealized with 0.1% Triton-XlOO, and blocked with 10% normal goat serum. Cells were then stained with the primary anti-CAR antibody (normal mouse IgG was used as a control) for 1 h followed by incubation with the AlexaFluor 594-labelled anti-mouse secondary antibody (Molecular Probes). 2.10. Membrane cholesterol measurement Membrane fractionation was performed as previously described (Yuan et al., 2005). The membrane cholesterol levels were measured using an Amplex Red Cholesterol Assay Kit according to the manufacturer’s instructions (Molecular Probes) and normalized to cell numbers. Briefly, membrane fractions dissolved in equal volume of solubilizing buffer were incubated with Amplex Red reaction mixture for 30 min at 37 ◦ C, and analyzed using a fluorometer with an excitation filter of 530 nm and an emission filter of 590 nm. 2.11. Statistical analysis The results are expressed as means ± standard errors (S.E.). Statistical analysis was performed with unpaired Student’s ttest. P-values less than 0.05 were considered to be statistically significant.
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3. Results 3.1. Lipofectamine-mediated transient transfection inhibits CVB3 infectivity Preliminary observations from our laboratory showed that transiently transfected HeLa cells were resistant to CVB3 infection, as reflected by the suppression of virus-induced cytopathic effect (Fig. 1A). To further investigate the effects of lipofectamine-mediated transient transfection on viral replication, we measured virus attachment, viral RNA and protein production, as well as virus titres by virus binding assay, in situ hybridization, Western blot, and plaque assay, respectively. Using lipofectamine, HeLa cells were transiently transfected for 48 h with various empty vectors, which were under the control of different promoters. Cells were then infected with CVB3 (MOI = 10) for 16 h and supernatant was collected and plaque assay was performed. Fig. 1B showed a significant reduction in viral progeny titres in transfected cells as compared to control cells. We also studied the effect of lipofectamine-mediated transient transfection on viral RNA replication and protein synthesis. In situ hybridization was performed to assess RNA expression on transfected HeLa cells at 5 h post-infection using a CVB3 antisense riboprobe. Viral RNA expression is drastically reduced in pcDNA- or pCMV-transfected cells as compared to control cells (Fig. 1C). Western blot was performed to examine viral protein expression. Eight hours post-infection, cell lysates were harvested and a mouse monoclonal antibody against CVB3 structural protein VP1 was used to measure viral protein expression. As shown in Fig. 1D, viral protein expression was significantly reduced in transfected cells. We further determined whether the reduction of viral RNA and protein synthesis was due to decreased virus attachment. Transiently transfected HeLa cells were infected with [35 S]labeled CVB3 for 1 h. The amount of radioisotope bound virus was measured using a scintillation counter. We showed that virus binding was significantly reduced in transfected cells as compared to non-transfected cells (Fig. 1E), suggesting that lipofectamine-mediated transient transfection reduces CVB3 infectivity likely through inhibition of virus attachment. It was noted that, in transfected cells, virus binding was reduced by approximately 60% (Fig. 1F), whereas virus titres and protein expression lowered by about 90% (Fig. 1B and D). We speculate that liposome-mediated transfection also inhibits viral internalization, which contributes to the observed discrepancy between the inhibitory effects of liposome-mediated transfection on virus binding and on viral protein expression as well as virus titres. Future study is needed to confirm this postulation. We also demonstrated that such viral inhibitory effects were observed in other cell lines, including HL-1 (murine atrial cardiomyocytes) and HEp2 cells (human larynx carcinoma cells) when they were transfected using different cationic lipids, including lipofectamine and effectene (Qiagen) (data not shown), indicating a general inhibition effect of liposomemediated transient tranfection on CVB3 infection.
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Fig. 1. Lipofectamine-mediated transient transfection inhibits CVB3 infectivity. HeLa cells were transiently transfected with various empty DNA vectors which are under the control of different promoters using lipofectamine following the manufacture’s guidelines. At 48 h post-transfection, cells were infected with CVB3 (MOI = 10). (A) Representative phase contrast images of transiently transfected HeLa cells at 8 h post-infection (pi). (B) Medium was collected from infected cells at 16 h pi and virus titers were determined by plaque assays on HeLa cell monolayers and were normalized to the protein concentrations of extracted HeLa cells. Values are means ± S.E. from three independent experiments, in each of which titrations were carried out in triplicate. (C) HeLa cells were fixed at 5 h pi and probed for viral RNA by in situ hybridization using antisense riboprobe (red). The cell nuclei were counterstained in blue by hematoxylin. (D) Cell lysates were collected at 8 h pi. CVB3 protein expression was analyzed by Western blotting using an anti-VPl antibody. To verify equal loading, the same blots were stripped and reprobed for -actin. (E) HeLa cells were transiently transfected as described above, then infected with [35 S]-labeled CVB3 (MOI = 1) for 1 h. Cell lysates were collected and measured for radioactivity by a scintillation counter (mean ± S.E., n = 3).
3.2. Lipoplex inhibits CVB3 infectivity To determine whether the viral inhibitory effect of transient transfection is intrinsic to transfection reagents, we applied lipofectamine and DNA individually, or as a complex to HeLa cells. Following 48 h incubation at 37 ◦ C, HeLa cells were infected
by CVB3. As shown in Fig. 2A, separate application of lipofectamine and DNA did not inhibit CVB3 infectivity. These data indicate that the formation of lipofectamine–DNA complex is crucial for its viral inhibitory effect. We also examined the influence of different doses of lipofectamine or DNA on viral replication. We found that the degree
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we examined the expression and localization of CVB3 receptor CAR by immunocytochemical staining. As shown in Fig. 3, CAR was exclusively localized to the plasma membrane in both control and transfected cells. There were no noticeable differences in the pattern of CAR localization and expression between two groups, suggesting that the reduced CVB3 infectivity after transient transfection is unlikely associated with the alteration of CAR expression and localization. 3.4. Addition of cholesterol reverses the viral inhibitory effect of lipofectamine-mediated transient transfection
Fig. 2. Lipofectamine–DNA complex inhibits CVB3 replication. (A) HeLa cells were treated with lipofectamine (6 l) or pcDNA vector (1.5 g) alone, or lipofectamine–DNA complex, followed by CVB3 infection, in an identical manner as described in Fig. 1. (B) HeLa cells were tranfected with different amount of pcDNA in the presence of 6 l lipofectamine (top panel) or with different amount of lipofectamine in the presence of 1.5 g pcDNA (lower panel) for 48 h, and then infected with CVB3. At 8 h pi, cell lysates were collected for Western blot analysis of VP1 expression. The same blots were reprobed for an antibody against -actin to illustrate equal protein loading.
of viral inhibition of lipoplex was dependent on the amount of lipofectamine and plasmid DNA (Fig. 2B). Furthermore, the viral inhibitory effect of transient transfection was maintained for 96 h post-transfection (data not shown). 3.3. Receptor localization after transient transfection To further understand the mechanisms by which lipofectamine-mediated transient transfection blocks viral infectivity,
To examine whether viral inhibition by lipofectaminemediated transient transfection results from membrane cholesterol depletion or disruption, cholesterol was applied together with lipoplex during transient transfection of HeLa cells. As shown in Fig. 4A, addition of cholesterol partially resumed VP1 expression in transfected cells, suggesting that inhibition of viral infectivity by lipoplex is due, at least in part, to changes of membrane cholesterol integrity. It was also noted that cholesterol application did not affect transfection efficiency as determined by -galactosidase activity assay (Fig. 4B). To determine whether the viral inhibitory effect by liposome– DNA complex is a general phenomenon for cholesterol-dependent viruses, we also studied respiratory syncytial virus (RSV), a known cholesterol-dependent virus (Gower and Graham, 2001; McCurdy and Graham, 2003). Here, we found that RSV infection was inhibited in HEp2 cells transiently transfected with various DNA vectors using Lipofectamine (data not shown), suggesting a general inhibition on the infection of cholesteroldependent viruses. 3.5. Lipofectamine-mediated transient transfection does not change membrane cholesterol levels To further determine the influences of lipofectaminemediated transient transfection on membrane cholesterol contents, we measured cholesterol levels at different times posttransfection. However, our data showed no significant difference in the total levels of membrane cholesterol between transfected and non-transfected groups (Fig. 5), implicating that disruption/sequestration rather than depletion of membrane choles-
Fig. 3. Localization of CAR in HeLa cells after lipofectamine-mediated transient transfection. HeLa cells were transiently transfected with pcDNA construct. Forty-eight hours later, cells were fixed and immunocytochemically stained for CAR.
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Fig. 5. Lipofectamine-mediated transient transfection does not change membrane cholesterol levels. HeLa cells were transiently transfected with pcDNA vector by lipofectamine. At 3 and 48 h post-transfection, cell lysates were collected and membrane fractionation was performed. Cholesterol levels were measured using an Amplex Red cholesterol assay kit and were normalized to the cell numbers. The data are expressed as a percentage of cholesterol in control untreated cells. Results are means ± S.E. (n = 6).
Fig. 4. Cholesterol replenishment reverses the inhibitory effect of lipofectaminemediated transient transfection on CVB3 replication. (A) Exogenous cholesterol at a concentration of 0.5 mM was applied together with lipoplex to HeLa cells during transient transfection. At 48 h post-transfection, HeLa cells were infected with CVB3. Six hours pi, cells were harvested and Western blot analysis was performed against VP1 expression. The same blots were reprobed for -actin to illustrate protein loading. VP1 expression was analyzed as described above (mean ± S.E., n = 3), and normalized to CVB3-infected cells alone (with cholesterol, but without transfection), which was arbitrarily set to a value of 1.0. (B) HeLa cells were transiently transfected with pcDNA-LacZ in the presence or absence of exogenous cholesterol (0.5 mM). Forty-eight hours later, cell lysates were collected and transfection efficiency was determined by -galactosidase activity assay.
terol contributes to the inhibitory effects of lipoplex on viral infectivity. 4. Discussion Cationic liposome-mediated gene delivery has been widely used for both the basic study of gene function and the clin-
ical application of gene therapy. The major advantages over viral gene transfer and traditional physiochemical gene delivery include its low immunogenecity, low toxicity, and ease of preparation (Zabner, 1997). Despite its wide use and potential applications in gene therapy, the influences of cationic lipidsmediated transfection on cellular function and the mechanisms involved are not well understood. In this study, we showed that application of lipoplex resulted in a strong inhibitory effect against CVB3 infectivity. Such inhibiting activity was dependent on the amount of transfection reagent and plasmid DNA applied and lasted at least for 96 h post-transfection. This inhibition was observed with lipoplex but not with the individual components of the complex. Furthermore, we found that cholesterol treatment partially restored the infection capacity of CVB3 inhibited by lipoplex. Finally, we showed that decreased viral infectivity appeared to be caused by the perturbation of membrane cholesterol integrity, rather than by cholesterol depletion. Taken together, our observations suggest for the first time that lipofectamine-mediated transient transfection inhibits CVB3 infectivity via inhibition of virus attachment, which is likely occurring through the disruption of membrane cholesterol integrity. Liposome–DNA complexes have been reported to prevent tumor cell growth and influence cancer metastasis capability. In cultured tumor cells, liposome–plasmid DNA complexes themselves were capable of inhibiting human ovarian carcinoma cell proliferation. Similar to our findings, such inhibition was independent of the target DNA sequence (Hofland and Huang, 1995). In mouse tumor models, systemic or local administration of liposome–DNA (empty vector) complexes induced a dramatic growth inhibition of various primary and metastatic tumors (Higgins et al., 2004; Hofland and Huang, 1995; Siders et al., 2002). It was further shown that the inhibitory effect of liposome–DNA complexes was significantly higher compared to “naked” DNA or cationic lipid reagent alone (Higgins et al., 2004; Hofland and Huang, 1995). Although the precise mechanisms of tumor inhibition by liposome–DNA complexes are
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still unclear, recent studies suggest that it may be dependent on their anti-proliferation and pro-inflammatory activities. In the present study, we showed that the application of lipoplex on HeLa cells reduced CVB3 infectivity. We and others have previously reported that CVB3 infection led to a blockage of cell cycle progression from Gl to S phase and that growth arrested cells produce higher levels of virus (Feuer et al., 2002; Luo et al., 2003). Therefore, the inhibitory effect of lipoplexes on CVB3 replication is unlikely to be due to their anti-proliferation activities. Lipid rafts have been known to play an important role in many biological processes, such as intracellular trafficking, signal transduction, and endo/exocytosis (Alonso and Millan, 2001; Simons and Toomre, 2000). Recently, involvement of lipidrafts in viral binding, viral entry, particle assembly and budding from the plasma membrane has also been recognized (see ref. Chazal and Gerlier, 2003, for a review). There is increasing evidence that many different viruses have evolved to use the host cell lipid rafts to support their replication. Although the role of lipid rafts in CVB3 infection has not yet been reported, known CVB3 receptors, CAR and DAF, can both associate with lipid rafts, suggesting a critical role of lipid rafts. Indeed, several closely related viruses of CVB3, such as echovirus (Stuart et al., 2002), coxsackievirus A9 (Triantafilou and Triantafilou, 2003), poliovirus (Danthi and Chow, 2004) and CVB4 (Triantafilou and Triantafilou, 2004), have been recently shown to utilize lipid raft microdomains or cholesterol, an important component of lipid rafts, to gain access to susceptible cells. To explore the potential mechanisms by which lipofectaminemediated transfection inhibits CVB3 infectivity, we applied exogenous cholesterol. We found that the capacity of CVB3 infection was partially restored after replenishment of cholesterol. Interestingly, we showed here that membrane cholesterol levels were unchanged during transfection, implying that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Indeed, studies have suggested that sequestering cholesterol out of lipid rafts by raftdisrupting reagents, such as nystatin and filipin, has similar effects on binding and entry of many viruses, including coxsackieviruses, as those of depleting cholesterol (Aizaki et al., 2004; Guyader et al., 2002; Nomura et al., 2004; Triantafilou and Triantafilou, 2003; Triantafilou and Triantafilou, 2004). The precise mechanisms of gene delivery by cationic lipids and the influence of transfection on cellular function are still unclear. It was initially thought that cationic liposome delivers DNA by fusion with the plasma membrane (Noguchi et al., 1998). However, increasing evidence suggests that liposome–DNA complexes enter host cells by an endocytic process, occurring through the cholesterol-dependent clathrin-mediated pathway (Zuhorn et al., 2002). Here, we speculate that endocytosis of lipoplex leads to a disruption or redistribution of the membrane cholesterol and addition of cholesterol reconstitutes the integrity of membrane cholesterol or reestablishes the association of membrane cholesterol with lipid rafts. In summary, we have shown that liposome-mediated transient transfection inhibits coxsackievirus B3 infectivity through
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reduction of viral attachment. This viral inhibition is independent of gene/vector property. We further show that viral inhibitory effect of lipoplex is associated with membrane cholesterol status or integrity. These studies provide new insights into our understanding of the initial virus–host interaction and suggest that liposome-mediated transient transfection of cDNA appear not to be a suitable approach to study the gene function in CVB3 infection. Acknowledgments This work was funded in part by grants from the Canadian Institutes of Health Research (CIHR) (HL), the Heart and Stroke Foundation of British Columbia and Yukon (HSFBCY) (HL), and the Canada Foundation for Innovation (HL). HL is a New Investigator of the CIHR/St. Paul’s Hospital Foundation Award and a Michael Smith Foundation for Health Research (MSFHR) Scholar. GG and ME are the recipients of a Doctoral Traineeship from the MSFHR and from the HSFBCY. XS is supported by a CIHR IMPACT Post-Doctoral Fellowship. YW is supported by a Fellowship from China Scholarship Council. References Aizaki, H., Lee, K.J., Sung, V.M., Ishiko, H., Lai, M.M., 2004. Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324, 450–461. Alonso, M.A., Millan, J., 2001. The role of lipid rafts in signalling and membrane trafficking in T lymphocytes. J. Cell Sci. 114, 3957–3965. Ashbourne Excoffon, K.J., Moninger, T., Zabner, J., 2003. The coxsackie B virus and adenovirus receptor resides in a distinct membrane microdomain. J. Virol. 77, 2559–2567. Barman, S., Adhikary, L., Chakrabarti, A.K., Bernas, C., Kawaoka, Y., Nayak, D.P., 2004. Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding. J. Virol. 78, 5258–5269. Bavari, S., Bosio, C.M., Wiegand, E., Ruthel, G., Will, A.B., Geisbert, T.W., Hevey, M., Schmaljohn, C., Schmaljohn, A., Aman, M.J., 2002. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195, 593–602. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.W., 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320–1323. Campbell, S.M., Crowe, S.M., Mak, J., 2001. Lipid rafts and HIV-1: from viral entry to assembly of progeny virions. J. Clin. Virol. 22, 217–227. Chazal, N., Gerlier, D., 2003. Virus entry, assembly, budding, and membrane rafts. Microbiol. Mol. Biol. Rev. 67, 226–237. Clements, G.B., Galbraith, D.N., Taylor, K.W., 1995. Coxsackie B virus infection and onset of childhood diabetes. Lancet 346, 221–223. Cohen, C.J., Shieh, J.T., Pickles, R.J., Okegawa, T., Hsieh, J.T., Bergelson, J.M., 2001. The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. Proc. Natl. Acad. Sci. U.S.A. 98, 15191–15196. Cuadras, M.A., Greenberg, H.B., 2003. Rotavirus infectious particles use lipid rafts during replication for transport to the cell surface in vitro and in vivo. Virology 313, 308–321. Danthi, P., Chow, M., 2004. Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry. J. Virol. 78, 33–41. Esfandiarei, M., Luo, H., Yanagawa, B., Suarez, A., Dabiri, D., Zhang, J., McManus, B.M., 2004. Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent. J. Virol. 78, 4289–4298.
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Noguchi, A., Furuno, T., Kawaura, C., Nakanishi, M., 1998. Membrane fusion plays an important role in gene transfection mediated by cationic liposomes. FEBS Lett. 433, 169–173. Nomura, R., Kiyota, A., Suzaki, E., Kataoka, K., Ohe, Y., Miyamoto, K., Senda, T., Fujimoto, T., 2004. Human coronavirus 229E binds to CD 13 in rafts and enters the cell through caveolae. J. Virol. 78, 8701– 8708. Parton, R.G., Richards, A.A., 2003. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4, 724– 738. Shafren, D.R., Bates, R.C., Agrez, M.V., Herd, R.L., Burns, G.F., Barry, R.D., 1995. Coxsackieviruses Bl, B3, and B5 use decay accelerating factor as a receptor for cell attachment. J. Virol. 69, 3873–3877. Siders, W.M., Vergillis, K., Johnson, C., Scheule, R.K., Kaplan, J.M., 2002. Tumor treatment with complexes of cationic lipid and noncoding plasmid DNA results in the induction of cytotoxic T cells and systemic tumor elimination. Mol. Ther. 6, 519–527. Simons, K., Ehehalt, R., 2002. Cholesterol, lipid rafts, and disease. J. Clin. Invest. 110, 597–603. Simons, K., Toomre, D., 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39. Stuart, A.D., Eustace, H.E., McKee, T.A., Brown, T.D., 2002. A novel cell entry pathway for a DAF-using human enterovirus is dependent on lipid rafts. J. Virol. 76, 9307–9322. Triantafilou, K., Triantafilou, M., 2003. Lipid raft microdomains: key sites for Coxsackievirus A9 infectious cycle. Virology 317, 128–135. Triantafilou, K., Triantafilou, M., 2004. Lipid-raft-dependent Coxsackievirus B4 internalization and rapid targeting to the Golgi. Virology 326, 6–19. Woodruff, J.F., 1980. Viral myocarditis. A review. Am. J. Pathol. 101, 425–484. Yuan, J., Zhang, J., Wong, B.W., Si, X., Wong, J., Yang, D., Luo, H., 2005. Inhibition of glycogen synthase kinase 3beta suppresses coxsackievirusinduced cytopathic effect and apoptosis via stabilization of beta-catenin. Cell Death Differ. 12, 1097–1106. Zabner, J., 1997. Cationic lipids used in gene transfer. Adv. Drug Deliv. Rev. 27, 17–28. Zuhorn, I.S., Kalicharan, R., Hoekstra, D., 2002. Lipoplex-mediated transfection of mammalian cells occurs through the cholesteroldependent clathrin-mediated pathway of endocytosis. J. Biol. Chem. 277, 18021–18028.
Liposome-mediated transient transfection reduces cholesterol-dependent coxsackievirus infectivity Jerry Wong, Jingchun Zhang, Guang Gao, Mitra Esfandiarei, Xiaoning Si, Yahong Wang, Bobby Yanagawa, Agripina Suarez, Bruce McManus, Honglin Luo ∗ Department of Pathology and Laboratory Medicine, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul’s Hospital, Vancouver, BC, Canada Received 11 September 2005; received in revised form 11 November 2005; accepted 15 November 2005 Available online 20 December 2005
Abstract Liposome-mediated gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Coxsackievirus B3 is an important human pathogen associated with various diseases. Here we reported that liposome-mediated transient transfection of plasmid cDNA inhibited coxsackieviral replication at the levels of RNA, protein and viral progeny release. These inhibitory effects were observed in various cell types and by using different liposome reagents. We further showed that the inhibition was likely due to the lack of virus attachment. Moreover, we showed that addition of cholesterol restored, at least in part, the viral infectivity. Interestingly, we found that membrane cholesterol levels were unchanged during transfection, indicating that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Our data suggest that liposome-mediated cDNA transient transfection inhibits coxsackievirus infectivity via inhibition of viral attachment, which is likely occurring through the changes of membrane cholesterol integrity. © 2005 Elsevier B.V. All rights reserved. Keywords: Liposome; Transient transfection; Coxsackievirus; Cholesterol; Lipid raft; Viral binding
1. Introduction Coxsackievirus B3 (CVB3) is a small, non-enveloped, positive-stranded virus belonging to the Enterovirus genus in the family of Picornaviridae. CVB3 is a common human pathogen associated with a broad spectrum of clinically relevant diseases including acute and chronic myocarditis, meningitis, pancreatitis and possibly insulin-dependent diabetes (Clements et al., 1995; McManus et al., 1991; Woodruff, 1980). Despite extensive efforts, knowledge regarding the mechanisms involved in coxsackievirus B3 infectivity, in particular the initial phase of viral–host interaction including viral attachment and internalization, is limited. The coxsackie-adenovirus receptor (CAR) (Bergelson et al., 1997; Martino et al., 2000) and the decay accelerating factor ∗
Corresponding author. The James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul’s Hospital, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6. Tel.: +1 604 682 2344x62847; fax: +1 604 806 8351. E-mail address: [email protected] (H. Luo). 0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.11.013
(DAF/CD55) (Martino et al., 1998; Shafren et al., 1995) have been identified as receptors for CVB3, which together facilitate cell binding and internalization of CVB3 into permissive host cells. CAR is a member of the junction adhesion molecule subgroup of the immunoglobulin superfamily (Cohen et al., 2001). Expression of CAR is highly regulated and generally correlated with viral tropism. DAF is a glycosylphosphatidylinositol (GPI)anchored protein, serving as a coreceptor for CVB3 infection. Recent studies have indicated that both CAR and DAF are localized to lipid-rich microdomains or lipid rafts of cell membranes, implicating that lipid rafts may be important for CVB3 attachment and entry (Ashbourne Excoffon et al., 2003; Stuart et al., 2002). Involvement of lipid rafts in viral replication has been reported in both enveloped viruses (human immunodeficiency virus (Campbell et al., 2001), influenza virus (Barman et al., 2004), Ebola virus (Bavari et al., 2002)), and non-enveloped viruses (simian virus 40 (Parton and Richards, 2003), echovirus 11 (Stuart et al., 2002), rotavirus (Cuadras and Greenberg, 2003), CVB4 (Triantafilou and Triantafilou, 2004), and coxsackievirus A9 (Triantafilou and Triantafilou, 2003)). Cholesterol is a critical component in lipid rafts which are involved in many cellular
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processes such as endo/exocytosis, intracellular trafficking, and membrane signaling (Alonso and Millan, 2001; Simons and Ehehalt, 2002). Gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Thus far, many techniques have been developed to deliver genes of interest into mammalian cells, which include viral gene transfer, electroporation, calcium phosphate co-precipitation, cationic lipids/DNA complexes, diethylaminoethyl-dextran/DNA, and polycation/dimethyl sulfoxide. Among them, cationic liposome-based transfection has been widely used for in vitro and in vivo gene delivery in eukaryotic cells due to the non-immunogenic and non-oncogenic properties, low cytotoxicity, and relatively high transfection efficiency (Zabner, 1997). The cellular and molecular mechanisms of cationic lipid-mediated gene transfer remains unclear. Early data suggested that cationic lipids-DNA complex (lipoplex) entered the cells by fusing with the cellular membrane (Noguchi et al., 1998). However, recent studies suggest that cationic liposome-mediated transfection of mammalian cells involves in an endocytotic process, occurring through a cholesteroldependent clathrin-mediated pathway (Zabner, 1997; Zuhorn et al., 2002). In a preliminary study, we observed a novel phenomenon that lipofectamine-mediated delivery of DNA inhibits CVB3 infectivity, which is independent of the properties of the vectors and genes. In this study, we further explore the viral inhibitory effect of cationic liposome-mediated transient transfection and attempt to define the mechanisms involved. We showed that CVB3 infectivity was reduced in HeLa cells which were transiently transfected by liposome. We further demonstrated that liposome–DNA complex suppression of viral infectivity was due, at least in part, to changes in the cellular cholesterol environment. 2. Materials and methods
of cells the next day using LipofectamineTM (Life Technologies) according to the manufacturer’s instructions. Briefly, lipofectamine (6 l) and DNA (1.5 g) were diluted in 100 l of Opti-MEM followed by equilibration at room temperature for 5–10 min after mixing. The lipofectamine–DNA complex was added to HeLa cells and incubated for 6 h. Cells were then washed with PBS and replenished with DMEM containing 10% serum. To examine transfection efficiency, cells transfected with a pcDNA-LacZ plasmid were lysed to determine the -galactosidase activity using a Promega kit according to the manufacturer’s recommendation. 2.3. Virus infection CVB3 (Nancy strain), a gift from Dr. Reinhard Kandolf (University Hospital of Tubingen, Tubingen, Germany), was propagated in HeLa cells and stored at −80 ◦ C. Virus titres were routinely determined by plaque assay on HeLa cell monolayers at the beginning of the experiments as described below. Cells were infected either with CVB3 at a multiplicity of infection (MOI) of 10 or with PBS (Sham). Following 1 h of incubation at 37 ◦ C, cells were washed with PBS and replenished with DMEM containing 10% serum. 2.4. Plaque assay The amount of CVB3 produced was measured on monolayers of HeLa cells by agar overlay plaque assay of supernatant cultures as previously described (Luo et al., 2002). Briefly, cell supernatant was serially diluted and overlaid on monolayers of HeLa cells. Following 1 h of incubation, medium was removed and complete DMEM containing 0.75% agar was overlaid. Three days post-infection, cells were fixed with Carnoy’s fixative (25% acetic acid, 75% ethanol) and then stained with 1% crystal violet. Viral titres were determined as plaque-forming units (PFU) per milliliter.
2.1. Cell culture and materials 2.5. Western blot analysis HeLa cells (HeLa S3) were obtained from the American Type Culture Collection. Subconfluent cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated newborn calf serum (Life Technologies) at 37 ◦ C in a humidified incubator with 5% CO2. Penicillin G (100 g/ml) and streptomycin (100 g/ml) (Life Technologies) were added to all culture media. Most supplies were purchased from Sigma Chemical Co. unless otherwise specified. The monoclonal antibody against CVB3 capsid protein 1 (VP1) was obtained from DakoCytomation. Monoclonal B-actin antibody, horseradish peroxidaseconjugated anti-rabbit and anti-mouse immunoglobulin G were purchased from Santa Cruz Biotechnology. The monoclonal anti-CAR antibody was from Upstate.
Western blot was performed as previously described (Luo et al., 2003). Briefly, equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech). The membrane was blocked with 5% non-fat dry milk solution containing 0.1% Tween-20 for 1 h. Afterwards, the membrane was incubated with primary antibody for 1 h, followed by secondary antibody for 45 min at room temperature. The immunoblots were visualized using an enhanced chemiluminescence detection system according to the manufacturer’s protocol (Amersham Pharmacia Biotech). 2.6. In situ hybridization
2.2. Transient transfection HeLa cells were plated onto six-well plates overnight and transfections were carried out on cells at 70–80% confluence
In situ hybridization was performed as previously described (Esfandiarei et al., 2004). Briefly, fixed cells were hybridized with digoxignenin-labeled CVB3 antisense riboprobes, which
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were prepared from the full-length CVB3 cDNA using an in vitro transcription kit (Promega) according to the manufacturer’s instructions. Hybridized riboprobes were detected using an alkaline phosphatase-conjugated anti-digoxignenin antibody (Roche Diagnostics) and a color substrate Vector Red (Vector Laboratories). Cells were counter stained with hematoxylin. 2.7. Virus radiolabeling and purification Confluent HeLa cells were infected with CVB3 at a MOI of 10 in methionine-free medium. Three hours after infection, medium containing 19.4 MBq/ml (525 Ci/ml) of tran [35 S] label (MP Biomedicals) was added for an additional 8 h. HeLa cells and medium were harvested and then frozen and thawed three times. The cellular debris was spun down and the supernatant was collected and centrifuged at 45,000 rpm for 90 min. The pellet containing [35 S]-labeled CVB3 was resuspended in serum-free DMEM and virus titer was measured by plaque assay and aliquots were stored at −80 ◦ C. 2.8. Virus binding assay Confluent HeLa cells were incubated with [35 S]-labeled CVB3 at a MOI of 1 (0.37 cpm/pfu) at 4 ◦ C for 1 h. The cells were washed thoroughly with PBS and lysed with 2% SDS. The amount of [35 S]-labeled CVB3 in the samples was measured with a 1217 rackbeta liquid scintillation counter (LKB Wallac). 2.9. Immunocytochemical staining At 48 h after transient transfection, HeLa cells were fixed with 4% paraformaldehyde, permealized with 0.1% Triton-XlOO, and blocked with 10% normal goat serum. Cells were then stained with the primary anti-CAR antibody (normal mouse IgG was used as a control) for 1 h followed by incubation with the AlexaFluor 594-labelled anti-mouse secondary antibody (Molecular Probes). 2.10. Membrane cholesterol measurement Membrane fractionation was performed as previously described (Yuan et al., 2005). The membrane cholesterol levels were measured using an Amplex Red Cholesterol Assay Kit according to the manufacturer’s instructions (Molecular Probes) and normalized to cell numbers. Briefly, membrane fractions dissolved in equal volume of solubilizing buffer were incubated with Amplex Red reaction mixture for 30 min at 37 ◦ C, and analyzed using a fluorometer with an excitation filter of 530 nm and an emission filter of 590 nm. 2.11. Statistical analysis The results are expressed as means ± standard errors (S.E.). Statistical analysis was performed with unpaired Student’s ttest. P-values less than 0.05 were considered to be statistically significant.
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3. Results 3.1. Lipofectamine-mediated transient transfection inhibits CVB3 infectivity Preliminary observations from our laboratory showed that transiently transfected HeLa cells were resistant to CVB3 infection, as reflected by the suppression of virus-induced cytopathic effect (Fig. 1A). To further investigate the effects of lipofectamine-mediated transient transfection on viral replication, we measured virus attachment, viral RNA and protein production, as well as virus titres by virus binding assay, in situ hybridization, Western blot, and plaque assay, respectively. Using lipofectamine, HeLa cells were transiently transfected for 48 h with various empty vectors, which were under the control of different promoters. Cells were then infected with CVB3 (MOI = 10) for 16 h and supernatant was collected and plaque assay was performed. Fig. 1B showed a significant reduction in viral progeny titres in transfected cells as compared to control cells. We also studied the effect of lipofectamine-mediated transient transfection on viral RNA replication and protein synthesis. In situ hybridization was performed to assess RNA expression on transfected HeLa cells at 5 h post-infection using a CVB3 antisense riboprobe. Viral RNA expression is drastically reduced in pcDNA- or pCMV-transfected cells as compared to control cells (Fig. 1C). Western blot was performed to examine viral protein expression. Eight hours post-infection, cell lysates were harvested and a mouse monoclonal antibody against CVB3 structural protein VP1 was used to measure viral protein expression. As shown in Fig. 1D, viral protein expression was significantly reduced in transfected cells. We further determined whether the reduction of viral RNA and protein synthesis was due to decreased virus attachment. Transiently transfected HeLa cells were infected with [35 S]labeled CVB3 for 1 h. The amount of radioisotope bound virus was measured using a scintillation counter. We showed that virus binding was significantly reduced in transfected cells as compared to non-transfected cells (Fig. 1E), suggesting that lipofectamine-mediated transient transfection reduces CVB3 infectivity likely through inhibition of virus attachment. It was noted that, in transfected cells, virus binding was reduced by approximately 60% (Fig. 1F), whereas virus titres and protein expression lowered by about 90% (Fig. 1B and D). We speculate that liposome-mediated transfection also inhibits viral internalization, which contributes to the observed discrepancy between the inhibitory effects of liposome-mediated transfection on virus binding and on viral protein expression as well as virus titres. Future study is needed to confirm this postulation. We also demonstrated that such viral inhibitory effects were observed in other cell lines, including HL-1 (murine atrial cardiomyocytes) and HEp2 cells (human larynx carcinoma cells) when they were transfected using different cationic lipids, including lipofectamine and effectene (Qiagen) (data not shown), indicating a general inhibition effect of liposomemediated transient tranfection on CVB3 infection.
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Fig. 1. Lipofectamine-mediated transient transfection inhibits CVB3 infectivity. HeLa cells were transiently transfected with various empty DNA vectors which are under the control of different promoters using lipofectamine following the manufacture’s guidelines. At 48 h post-transfection, cells were infected with CVB3 (MOI = 10). (A) Representative phase contrast images of transiently transfected HeLa cells at 8 h post-infection (pi). (B) Medium was collected from infected cells at 16 h pi and virus titers were determined by plaque assays on HeLa cell monolayers and were normalized to the protein concentrations of extracted HeLa cells. Values are means ± S.E. from three independent experiments, in each of which titrations were carried out in triplicate. (C) HeLa cells were fixed at 5 h pi and probed for viral RNA by in situ hybridization using antisense riboprobe (red). The cell nuclei were counterstained in blue by hematoxylin. (D) Cell lysates were collected at 8 h pi. CVB3 protein expression was analyzed by Western blotting using an anti-VPl antibody. To verify equal loading, the same blots were stripped and reprobed for -actin. (E) HeLa cells were transiently transfected as described above, then infected with [35 S]-labeled CVB3 (MOI = 1) for 1 h. Cell lysates were collected and measured for radioactivity by a scintillation counter (mean ± S.E., n = 3).
3.2. Lipoplex inhibits CVB3 infectivity To determine whether the viral inhibitory effect of transient transfection is intrinsic to transfection reagents, we applied lipofectamine and DNA individually, or as a complex to HeLa cells. Following 48 h incubation at 37 ◦ C, HeLa cells were infected
by CVB3. As shown in Fig. 2A, separate application of lipofectamine and DNA did not inhibit CVB3 infectivity. These data indicate that the formation of lipofectamine–DNA complex is crucial for its viral inhibitory effect. We also examined the influence of different doses of lipofectamine or DNA on viral replication. We found that the degree
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we examined the expression and localization of CVB3 receptor CAR by immunocytochemical staining. As shown in Fig. 3, CAR was exclusively localized to the plasma membrane in both control and transfected cells. There were no noticeable differences in the pattern of CAR localization and expression between two groups, suggesting that the reduced CVB3 infectivity after transient transfection is unlikely associated with the alteration of CAR expression and localization. 3.4. Addition of cholesterol reverses the viral inhibitory effect of lipofectamine-mediated transient transfection
Fig. 2. Lipofectamine–DNA complex inhibits CVB3 replication. (A) HeLa cells were treated with lipofectamine (6 l) or pcDNA vector (1.5 g) alone, or lipofectamine–DNA complex, followed by CVB3 infection, in an identical manner as described in Fig. 1. (B) HeLa cells were tranfected with different amount of pcDNA in the presence of 6 l lipofectamine (top panel) or with different amount of lipofectamine in the presence of 1.5 g pcDNA (lower panel) for 48 h, and then infected with CVB3. At 8 h pi, cell lysates were collected for Western blot analysis of VP1 expression. The same blots were reprobed for an antibody against -actin to illustrate equal protein loading.
of viral inhibition of lipoplex was dependent on the amount of lipofectamine and plasmid DNA (Fig. 2B). Furthermore, the viral inhibitory effect of transient transfection was maintained for 96 h post-transfection (data not shown). 3.3. Receptor localization after transient transfection To further understand the mechanisms by which lipofectamine-mediated transient transfection blocks viral infectivity,
To examine whether viral inhibition by lipofectaminemediated transient transfection results from membrane cholesterol depletion or disruption, cholesterol was applied together with lipoplex during transient transfection of HeLa cells. As shown in Fig. 4A, addition of cholesterol partially resumed VP1 expression in transfected cells, suggesting that inhibition of viral infectivity by lipoplex is due, at least in part, to changes of membrane cholesterol integrity. It was also noted that cholesterol application did not affect transfection efficiency as determined by -galactosidase activity assay (Fig. 4B). To determine whether the viral inhibitory effect by liposome– DNA complex is a general phenomenon for cholesterol-dependent viruses, we also studied respiratory syncytial virus (RSV), a known cholesterol-dependent virus (Gower and Graham, 2001; McCurdy and Graham, 2003). Here, we found that RSV infection was inhibited in HEp2 cells transiently transfected with various DNA vectors using Lipofectamine (data not shown), suggesting a general inhibition on the infection of cholesteroldependent viruses. 3.5. Lipofectamine-mediated transient transfection does not change membrane cholesterol levels To further determine the influences of lipofectaminemediated transient transfection on membrane cholesterol contents, we measured cholesterol levels at different times posttransfection. However, our data showed no significant difference in the total levels of membrane cholesterol between transfected and non-transfected groups (Fig. 5), implicating that disruption/sequestration rather than depletion of membrane choles-
Fig. 3. Localization of CAR in HeLa cells after lipofectamine-mediated transient transfection. HeLa cells were transiently transfected with pcDNA construct. Forty-eight hours later, cells were fixed and immunocytochemically stained for CAR.
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Fig. 5. Lipofectamine-mediated transient transfection does not change membrane cholesterol levels. HeLa cells were transiently transfected with pcDNA vector by lipofectamine. At 3 and 48 h post-transfection, cell lysates were collected and membrane fractionation was performed. Cholesterol levels were measured using an Amplex Red cholesterol assay kit and were normalized to the cell numbers. The data are expressed as a percentage of cholesterol in control untreated cells. Results are means ± S.E. (n = 6).
Fig. 4. Cholesterol replenishment reverses the inhibitory effect of lipofectaminemediated transient transfection on CVB3 replication. (A) Exogenous cholesterol at a concentration of 0.5 mM was applied together with lipoplex to HeLa cells during transient transfection. At 48 h post-transfection, HeLa cells were infected with CVB3. Six hours pi, cells were harvested and Western blot analysis was performed against VP1 expression. The same blots were reprobed for -actin to illustrate protein loading. VP1 expression was analyzed as described above (mean ± S.E., n = 3), and normalized to CVB3-infected cells alone (with cholesterol, but without transfection), which was arbitrarily set to a value of 1.0. (B) HeLa cells were transiently transfected with pcDNA-LacZ in the presence or absence of exogenous cholesterol (0.5 mM). Forty-eight hours later, cell lysates were collected and transfection efficiency was determined by -galactosidase activity assay.
terol contributes to the inhibitory effects of lipoplex on viral infectivity. 4. Discussion Cationic liposome-mediated gene delivery has been widely used for both the basic study of gene function and the clin-
ical application of gene therapy. The major advantages over viral gene transfer and traditional physiochemical gene delivery include its low immunogenecity, low toxicity, and ease of preparation (Zabner, 1997). Despite its wide use and potential applications in gene therapy, the influences of cationic lipidsmediated transfection on cellular function and the mechanisms involved are not well understood. In this study, we showed that application of lipoplex resulted in a strong inhibitory effect against CVB3 infectivity. Such inhibiting activity was dependent on the amount of transfection reagent and plasmid DNA applied and lasted at least for 96 h post-transfection. This inhibition was observed with lipoplex but not with the individual components of the complex. Furthermore, we found that cholesterol treatment partially restored the infection capacity of CVB3 inhibited by lipoplex. Finally, we showed that decreased viral infectivity appeared to be caused by the perturbation of membrane cholesterol integrity, rather than by cholesterol depletion. Taken together, our observations suggest for the first time that lipofectamine-mediated transient transfection inhibits CVB3 infectivity via inhibition of virus attachment, which is likely occurring through the disruption of membrane cholesterol integrity. Liposome–DNA complexes have been reported to prevent tumor cell growth and influence cancer metastasis capability. In cultured tumor cells, liposome–plasmid DNA complexes themselves were capable of inhibiting human ovarian carcinoma cell proliferation. Similar to our findings, such inhibition was independent of the target DNA sequence (Hofland and Huang, 1995). In mouse tumor models, systemic or local administration of liposome–DNA (empty vector) complexes induced a dramatic growth inhibition of various primary and metastatic tumors (Higgins et al., 2004; Hofland and Huang, 1995; Siders et al., 2002). It was further shown that the inhibitory effect of liposome–DNA complexes was significantly higher compared to “naked” DNA or cationic lipid reagent alone (Higgins et al., 2004; Hofland and Huang, 1995). Although the precise mechanisms of tumor inhibition by liposome–DNA complexes are
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still unclear, recent studies suggest that it may be dependent on their anti-proliferation and pro-inflammatory activities. In the present study, we showed that the application of lipoplex on HeLa cells reduced CVB3 infectivity. We and others have previously reported that CVB3 infection led to a blockage of cell cycle progression from Gl to S phase and that growth arrested cells produce higher levels of virus (Feuer et al., 2002; Luo et al., 2003). Therefore, the inhibitory effect of lipoplexes on CVB3 replication is unlikely to be due to their anti-proliferation activities. Lipid rafts have been known to play an important role in many biological processes, such as intracellular trafficking, signal transduction, and endo/exocytosis (Alonso and Millan, 2001; Simons and Toomre, 2000). Recently, involvement of lipidrafts in viral binding, viral entry, particle assembly and budding from the plasma membrane has also been recognized (see ref. Chazal and Gerlier, 2003, for a review). There is increasing evidence that many different viruses have evolved to use the host cell lipid rafts to support their replication. Although the role of lipid rafts in CVB3 infection has not yet been reported, known CVB3 receptors, CAR and DAF, can both associate with lipid rafts, suggesting a critical role of lipid rafts. Indeed, several closely related viruses of CVB3, such as echovirus (Stuart et al., 2002), coxsackievirus A9 (Triantafilou and Triantafilou, 2003), poliovirus (Danthi and Chow, 2004) and CVB4 (Triantafilou and Triantafilou, 2004), have been recently shown to utilize lipid raft microdomains or cholesterol, an important component of lipid rafts, to gain access to susceptible cells. To explore the potential mechanisms by which lipofectaminemediated transfection inhibits CVB3 infectivity, we applied exogenous cholesterol. We found that the capacity of CVB3 infection was partially restored after replenishment of cholesterol. Interestingly, we showed here that membrane cholesterol levels were unchanged during transfection, implying that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Indeed, studies have suggested that sequestering cholesterol out of lipid rafts by raftdisrupting reagents, such as nystatin and filipin, has similar effects on binding and entry of many viruses, including coxsackieviruses, as those of depleting cholesterol (Aizaki et al., 2004; Guyader et al., 2002; Nomura et al., 2004; Triantafilou and Triantafilou, 2003; Triantafilou and Triantafilou, 2004). The precise mechanisms of gene delivery by cationic lipids and the influence of transfection on cellular function are still unclear. It was initially thought that cationic liposome delivers DNA by fusion with the plasma membrane (Noguchi et al., 1998). However, increasing evidence suggests that liposome–DNA complexes enter host cells by an endocytic process, occurring through the cholesterol-dependent clathrin-mediated pathway (Zuhorn et al., 2002). Here, we speculate that endocytosis of lipoplex leads to a disruption or redistribution of the membrane cholesterol and addition of cholesterol reconstitutes the integrity of membrane cholesterol or reestablishes the association of membrane cholesterol with lipid rafts. In summary, we have shown that liposome-mediated transient transfection inhibits coxsackievirus B3 infectivity through
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reduction of viral attachment. This viral inhibition is independent of gene/vector property. We further show that viral inhibitory effect of lipoplex is associated with membrane cholesterol status or integrity. These studies provide new insights into our understanding of the initial virus–host interaction and suggest that liposome-mediated transient transfection of cDNA appear not to be a suitable approach to study the gene function in CVB3 infection. Acknowledgments This work was funded in part by grants from the Canadian Institutes of Health Research (CIHR) (HL), the Heart and Stroke Foundation of British Columbia and Yukon (HSFBCY) (HL), and the Canada Foundation for Innovation (HL). HL is a New Investigator of the CIHR/St. Paul’s Hospital Foundation Award and a Michael Smith Foundation for Health Research (MSFHR) Scholar. GG and ME are the recipients of a Doctoral Traineeship from the MSFHR and from the HSFBCY. XS is supported by a CIHR IMPACT Post-Doctoral Fellowship. YW is supported by a Fellowship from China Scholarship Council. References Aizaki, H., Lee, K.J., Sung, V.M., Ishiko, H., Lai, M.M., 2004. Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324, 450–461. Alonso, M.A., Millan, J., 2001. The role of lipid rafts in signalling and membrane trafficking in T lymphocytes. J. Cell Sci. 114, 3957–3965. Ashbourne Excoffon, K.J., Moninger, T., Zabner, J., 2003. The coxsackie B virus and adenovirus receptor resides in a distinct membrane microdomain. J. Virol. 77, 2559–2567. Barman, S., Adhikary, L., Chakrabarti, A.K., Bernas, C., Kawaoka, Y., Nayak, D.P., 2004. Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding. J. Virol. 78, 5258–5269. Bavari, S., Bosio, C.M., Wiegand, E., Ruthel, G., Will, A.B., Geisbert, T.W., Hevey, M., Schmaljohn, C., Schmaljohn, A., Aman, M.J., 2002. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195, 593–602. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.W., 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320–1323. Campbell, S.M., Crowe, S.M., Mak, J., 2001. Lipid rafts and HIV-1: from viral entry to assembly of progeny virions. J. Clin. Virol. 22, 217–227. Chazal, N., Gerlier, D., 2003. Virus entry, assembly, budding, and membrane rafts. Microbiol. Mol. Biol. Rev. 67, 226–237. Clements, G.B., Galbraith, D.N., Taylor, K.W., 1995. Coxsackie B virus infection and onset of childhood diabetes. Lancet 346, 221–223. Cohen, C.J., Shieh, J.T., Pickles, R.J., Okegawa, T., Hsieh, J.T., Bergelson, J.M., 2001. The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. Proc. Natl. Acad. Sci. U.S.A. 98, 15191–15196. Cuadras, M.A., Greenberg, H.B., 2003. Rotavirus infectious particles use lipid rafts during replication for transport to the cell surface in vitro and in vivo. Virology 313, 308–321. Danthi, P., Chow, M., 2004. Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry. J. Virol. 78, 33–41. Esfandiarei, M., Luo, H., Yanagawa, B., Suarez, A., Dabiri, D., Zhang, J., McManus, B.M., 2004. Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent. J. Virol. 78, 4289–4298.
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