Department of Microbiology, 5 Science Drive 2, National University of Singapore, 117597, Singapore1
Author for correspondence: M. L. Ng. Fax +65 776 6872. e-mail micngml{at}nus.edu.sg
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Abstract |
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Introduction |
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Polarized epithelial cells line the major cavities of the body and form a selective barrier against the invasion of many pathogens. The plasma membranes of these polarized cells are highly specialized and are divided into two discrete surfaces, the apical and the basolateral surface. The apical and basolateral domains of polarized cells each display a distinct set of specialized proteins, lipids and cellular receptors (Gumbiner & Simons, 1987 ; Madara, 1988
; Simons & Wandinger-Ness, 1990
). These discrete domains function in the selective absorption and release of many cellular proteins and pathogens. Polarized epithelial cells have been used extensively to study the sorting of cellular and viral proteins in polarized cells. Sorting cellular receptors and proteins to the distinct membrane domains of polarized cells has been reported to be mediated by the interaction of the cytoskeletal network (Wandinger-Ness & Simons, 1991
). Several studies over the years have investigated polarized entry, the intracellular sorting mechanism of viral proteins and the release of virus progeny from distinct domains of polarized cells (Tucker & Compans, 1993
; Compans, 1995
). These studies have provided crucial information on the distribution of virus cellular receptors, the intracellular trafficking mechanism of viral proteins during morphogenesis and the domains of virus egress. The polarized entry and release of viruses might also be significant for virus pathogenicity (reviewed by Compans, 1995
).
So far, the interaction of flaviviruses with polarized cells has not been documented. In this study, polarized entry and release of WN virus strain Sarafend (S) and KUN virus from specific domains of polarized epithelial cells (Vero C1008) were defined. Microtubule-dependent, polarized sorting of flavivirus E proteins in polarized Vero C1008 cells was also demonstrated.
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Methods |
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Reagents and antibodies.
Propidium iodide and vinblastine sulphate were purchased from Sigma. The antibody for the WN virus E protein was a monospecific antibody raised in rabbits and was a kind gift from Vincent Deubel, Pasteur Institute, France. The anti-KUN virus E protein antibody, produced in our laboratory, was a polyclonal antibody derived from rabbits. Anti-tubulin and secondary antibodies conjugated to either FITC or Texas red (TR) were purchased from Amersham Pharmacia.
Indirect immunofluorescence microscopy.
For immunofluorescence microscopy, cell monolayers were grown on cell inserts and infected with WN (S) or KUN virus at an m.o.i. of 10. At 12 and 24 h p.i. for WN (S) and KUN virus, respectively, the cells were fixed with cold absolute methanol for 10 min, followed by a wash in cold PBS for 15 min. The cells were then washed three times for 10 min each in cold PBS containing 0·1% BSA to eliminate non-specific binding. Cells were incubated with the primary antibodies [1:30 dilution, anti-WN (S) virus E protein antibody; 1:10 dilution, anti-KUN virus E protein antibody; 1:50 dilution, anti-tubulin antibody] in a humidity chamber for 1 h at 37 °C. After three washes for 5 min each with PBS, cells were incubated further with FITC- or TR-conjugated secondary antibodies before washing again with PBS. Virus-infected cells were also incubated for 10 min with propidium iodide at a concentration of 0·1 µg/ml. The processed cell inserts were mounted onto ethanol-cleaned glass slides using Dabco mountant. Specimens were viewed under a laser scanning confocal inverted microscope (Leica TCS SP2) and an optical microscope (Olympus, BX60).
Ultrastructural analysis.
Vero and Vero C1008 cells were grown until confluent on cell inserts and were then infected apically with either WN (S) or KUN virus at an m.o.i. of 10. At the end of the infection period, 12 h for WN (S) virus and 24 h for KUN virus, the infected cells grown on 0·4 µm porous membranes were washed twice with cold PBS and prefixed with a 2·5% glutaraldehyde2% paraformaldehyde mixture. This was followed by post-fixation for 90 min at 4 °C in 1% osmium tetroxide. The samples were then dehydrated through a series of ethanol solutions of increasing concentration and embedded in low-viscosity epoxy resin. Ultrathin sections (5070 nm) were stained with 2% uranyl acetate and post-stained with 2% lead citrate before viewing under a Philips electron microscope, CM 120 Biotwin.
Microtubule-disrupting drug treatment.
Polarized Vero C1008 cells grown on cell culture inserts were infected apically with WN (S) or KUN virus at an m.o.i. of 10. At 6 [WN (S) virus] and 12 (KUN virus) h p.i., culture media from both apical and basolateral chambers were supplemented with 10 µg/ml vinblastine sulphate (Sigma). The supernatants from both apical and basolateral chambers were then harvested at 12 h p.i. for WN (S) virus and 24 h p.i. for KUN virus. Titres of productive virus were determined by plaque assays and the infected cells on the culture inserts were also fixed and processed for immunofluorescence assays.
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Results |
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Hence, these results have provided strong evidence that entry of both WN (S) and KUN viruses was restricted to the apical surface of polarized Vero C1008 cells. This could suggest that the receptor molecules for both WN and KUN viruses were highly expressed at the apical domain of polarized epithelial cells.
Release of WN virus is restricted to the apical domain of Vero C1008 cells while KUN virus is released bi-directionally
To determine the release site of the flaviviruses used in this study, WN (S) or KUN virus was inoculated as described in Methods. At different time-points, infected supernatants from either the apical or the basolateral chambers were assessed for progeny virus by plaque assays. WN (S) virus was released predominantly from the apical surface of Vero C1008 cells (Fig. 3a). At 12 h p.i., virus titres of WN (S) virus from the apical surface were more than 4 log units higher than those at the basolateral surface. In contrast, bi-directional release of KUN virus was observed (Fig. 3b
). The titres of KUN virus released from the apical domain were approximately equivalent to those from the basolateral domain throughout the time-sequence study.
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To confirm the above results further, localization of virus E proteins and virus particles at certain domains of Vero C1008 cells during virus release was visualized using immunofluorescence coupled with confocal microscopy and transmission electron microscopy. The WN (S) virus E protein and probably virus particles were observed as yellowish speckles of fluorescence localized exclusively at the apical surface of the infected cells (Fig. 4a). In contrast, KUN virus was localized at both domains of the infected cell monolayer (Fig. 4b
).
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Polarized sorting of WN virus E protein to the apical domain of Vero C1008 cells is dependent on an intact microtubule network
In a recent study involving non-polarized Vero cells, the E and C proteins of WN (S) virus were transported along cellular microtubules from the site of synthesis (i.e. endoplasmic recticulum and Golgi apparatus) to the cell periphery for assembly (Ng et al., 2001 ; Chu & Ng, 2002
). However, it is not known whether microtubules are involved in the polarized sorting of WN (S) virus E protein in Vero C1008 cells; this could contribute to the apical release of WN (S) virus.
In these series of experiments, the possible involvement of microtubules in the polarized sorting of the E proteins of WN and KUN viruses was investigated. WN (S) or KUN virus-infected Vero C1008 cells were treated with a microtubule-disrupting drug, vinblastine sulphate, at a concentration of 10 µg/ml. At this concentration, vinblastine sulphate induces rapid depolymerization of the cellular microtubules but has minimal cytotoxic effects on Vero C1008 cells. Despite the formation of vinblastine sulphate-induced microtubulin paracrystals, minimal cell rounding and cell death was observed (data not shown).
As shown in Fig. 6, disruption of the microtubule network led to a drastic inhibition of the apical release of WN (S) virus but not KUN virus. However, the basolateral release of WN (S) virus was not affected when compared to that seen in Fig. 3(a)
. Furthermore, WN (S) and KUN virus-infected Vero cells treated with vinblastine sulphate were also processed for indirect immunofluorescence assay at 12 and 24 h p.i., respectively. In Fig. 7
, entrapment of WN (S) virus E protein (Fig. 7
, arrows) with the drug-induced microtubulin paracrystals (Fig. 7
, arrowheads) was observed. Yellow staining indicated co-localization of the E protein (FITC) with the microtubulin paracrystals (TR). Staining of WN virus E proteins was not observed in the cytoplasm and cell plasma membrane. In contrast, KUN virus E proteins were localized to the cell cytoplasm and plasma membrane but not the drug-induced microtubulin paracrystals (data not shown). Together these results strongly suggested that the intracellular sorting of WN (S) virus E protein to the apical surface is dependent on the microtubule network, whereas KUN virus is not.
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Discussion |
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Previous studies have shown that entry of WN (S) and KUN viruses was mediated by receptor-mediated endocytosis (Gollins & Porterfield, 1985 ; Kimura et al., 1986
; Ng & Lau, 1988
). However, the actual cellular receptors involved in the initial attachment and entry of these viruses have yet to be identified. In the present study, investigation revealed the polarized entry of WN (S) and KUN viruses into Vero C1008 cells. It was found that both WN (S) and KUN virus infections occurred with much higher efficiency when the viruses were inoculated at the apical surface of Vero C1008 cells (Fig. 1
). This type of restriction in the entry of viruses has also been documented for simian virus 40, measles virus, Black Creek Canal virus, EpsteinBarr virus and hepatitis A virus (Clayson & Compans, 1988
; Blau & Compans, 1995
; Chodash et al., 2000
; Blank et al., 2000
). For these viruses, polarized entry was aided by the apical localization of their cellular receptors in Vero C1008 cells. Since polarized epithelial cells express distinct sets of receptors and cellular proteins at either the apical or the basolateral domain, the preferential entry of WN (S) and KUN viruses could imply the apical localization of their cellular receptors in Vero C1008 cells. With the receptor-rich region identified in this cell line, it could be a good cell system for the isolation of the receptor molecule(s) in future studies.
Polarized release of viruses from polarized epithelial cells was first observed in enveloped RNA viruses. Influenza virions were observed to bud from the apical membrane, while vesicular stomatitis virus was released basolaterally (Rodriguez-Boulan et al., 1983 ). The polarized fashion of virus release was subsequently found to be attributed to the polarized expression and intracellular sorting of the respective viral proteins to either the apical or the basolateral surface for release (Boulan & Pendergast, 1980
; Matlin & Simons, 1984
; Misek et al., 1984
; Rindler et al., 1985
; Gottlieb et al., 1986
; Fuller et al., 1985
).
Several intracellular sorting mechanisms occurring in polarized cells have been proposed. One of these was shown to involve the active role of the cytoskeleton network (Wandinger-Ness & Simons, 1991 ). The cytoskeleton network, especially the microtubules, has been shown to play an important role in the polarized sorting of viral proteins to discrete domains of the polarized cells. Microtubule disrupting drugs, such as nocodazole, colchine and vinblastine sulphate have been used commonly to investigate the involvement of microtubules on the intracellular polarized sorting of cellular and viral proteins (Thyberg & Moskalewski, 1985
). Of the earlier studies, Eilers et al. (1989)
used the Caco-2 cell line (intestinal epithelial cells) to study the involvement of microtubules on the intracellular sorting mechanism of cell membrane and secreting proteins. These authors have shown convincingly that the delivery of these proteins to the apical domain of the polarized cells is impaired in the presence of microtubule-disrupting drugs.
In this study, disruption of the microtubule network with vinblastine sulphate has revealed that the sorting of WN virus E proteins to the apical domain of Vero C1008 cells was highly dependent on the presence of intact microtubules. A drastic decrease in the apical release of WN (S) virus was observed upon disruption of the microtubule network by vinblastine sulphate (compare Fig. 6 with Fig. 3a
). The reduction in the apical release of WN (S) virus was probably caused by the entrapment of the viral structural proteins with the depolymerized microtubules, which failed to deliver the viral structural proteins to the apical surface for virion assembly (Fig. 7
). Using a similar system, Bose et al. (2001)
have also recently demonstrated that the apical release of human parainfluenza virus type 3 was highly dependent on the presence of intact microtubules. The disruption of microtubules by nacodazole was shown to cause a drastic inhibition in the release of the virus from the apical domain.
As for KUN virus, the disruption of microtubules during late infection did not affect the direction of release and the titres of progeny virus (Fig. 6, compare with Fig. 3b
). Bi-directional release of KUN virus by exocytosis was observed in Vero C1008 cells (Figs 4b
and 5c
), in contrast to WN (S) virus. One possible explanation for the difference in the release of the closely related virus could be due to the different modes of maturation of the two viruses. The mode of maturation of KUN virus has been well documented. KUN virus matures intracellularly at the endoplasmic recticulum and the Golgi apparatus and mature virus particles are transported by a single vesicular transport step to the plasma membrane, independently of the microtubules. Virus particles are then released by exocytosis (Mackenzie & Westaway, 2001
).
However, Ng et al. (1994) documented an alternative mode of maturation of WN (S) virus. In contrast to the usual intracellular maturation of flaviviruses, WN (S) virus was observed to mature by budding at the plasma membrane. In addition, our recent study (Chu & Ng, 2002
) has demonstrated that the microtubule network is essential in the trafficking of WN (S) virus structural proteins to the plasma membrane for morphogenesis of the virus in non-polarized cells. Hence, the apical egress of WN (S) virus from polarized epithelial Vero C1008 cells was contributed to by the microtubule-dependent polarized sorting of the structural proteins to the apical membrane, while the intracellular delivery of mature KUN virus particles is independent of the microtubule network. Whether the differences in the polarized release of WN (S) and KUN viruses contribute to the pathogenesis of these viruses is currently under investigation.
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Acknowledgments |
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References |
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Received 14 January 2002;
accepted 17 June 2002.