Department of Virology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany
Correspondence
Beate Sodeik
Sodeik.Beate{at}MH-Hannover.de
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ABSTRACT |
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Present address: Max-von-Pettenkofer Institut, Ludwig-Maximilians-Universität, München, Germany.
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INTRODUCTION |
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Like many DNA viruses, HSV-1 has to deliver its genome of 152 kbp from the plasma membrane to the cell nucleus, where viral transcription and DNA replication take place (Roizman & Knipe, 2001; Whittaker et al., 2000
). The genome is packaged into a pre-assembled nuclear capsid with a diameter of 125 nm, which is then further coated with an amorphous protein layer called the tegument. To complete virus assembly, a lipid membrane, the virus envelope, is wrapped around the tegument (reviewed by Enquist et al., 1998
; Mettenleiter, 2002
).
The entry of HSV-1 into cells involves a series of interactions between several viral envelope proteins and molecules of the host plasma membrane. Infection is initiated by the attachment of HSV-1 glycoprotein C (gC) and gB to heparan or chondroitin sulfate proteoglycans (reviewed by (Spear et al., 2000). Nectin-1 (HveC) is considered to be the most important receptor for HSV-1 gD and entry into epithelial cells (Geraghty et al., 1998
). Additional HSV-1 gD receptors are HveA (Montgomery et al., 1996
) and a heparan sulfate modified by a 3-O-sulfotransferase (Shukla et al., 1999
). Nectin-1, like nectin-2, -3, -4 and the homologous poliovirus receptor, belong to the Ca2+-independent cell adhesion proteins of the immunoglobulin superfamily. Nectins mainly co-localize with the Ca2+-dependent cell adhesion protein E-cadherin and catenin in the adherens junctions of epithelial cells. Most forms of nectin can bind with their cytosolic tail to the PDZ domain of l-afadin, an actin-binding protein (Takai & Nakanishi, 2003
).
HSV-1 gD in combination with gB and the gH/gL complex trigger the fusion of the viral envelope with the cell membrane, which leads to the release of the tegument proteins and the viral capsid into the cytosol (Spear et al., 2000). While alphaherpesvirus infection is often mediated by pH-neutral fusion with the plasma membrane, certain cell types support an endocytic, pH-dependent route for HSV-1 entry (Nicola et al., 2003
).
After penetration of the actin cortex underneath the plasma membrane, the capsids are transported in many cell types along microtubules to the cell nucleus (Mabit et al., 2002; Sodeik et al., 1997
). Microtubules are hollow polar protein cylinders polymerized from
/
-tubulin heterodimers. In cultured cells, their so-called minus ends are usually fixed at a microtubule-organizing centre, which is often localized in close proximity to the cell nucleus. The plus ends of microtubules extend into the cell periphery (Döhner & Sodeik, 2004
). Like many host organelles, the capsids are transported in unpolarized epithelial cells by the minus-end directed microtubule-activated ATPase dynein and its cofactor dynactin to the microtubule-organizing centre in the cell centre (Döhner et al., 2002
; Sodeik et al., 1997
). The capsid binds via importin-
to the nuclear pore and this interaction triggers the release of the HSV-1 genome into the nucleoplasm and allows the onset of virus replication (Ojala et al., 2000
).
The cell lines MadinDarby canine kidney (MDCK) and Caco-2, a human Caucasian colon adenocarcinoma cell line, can be induced to differentiate and form an epithelial-like monolayer when grown on porous filters for several days (Rothen-Rutishauser et al., 1998; Rousset, 1986
; Simons & Virta, 1998
). For this reason, they have been extensively used as models to study epithelial polarization and virus infection of epithelial cells (Compans, 1995
). Previous studies have suggested that polarized MDCK and Caco-2 cells can be infected from both the apical and basolateral surface with HSV-1 or a replication-incompetent HSV-1 helper virus (Griffiths et al., 1998
; Hayashi, 1995
; Murphy et al., 1997
; Sears et al., 1991
; Topp et al., 1997
; Tran et al., 2000
).
In highly polarized epithelial cells, microtubules are usually arranged in an apicalbasal direction with their minus ends oriented towards the apical surface while the plus ends point towards the basal region of the cell (Grindstaff et al., 1998; Meads & Schroer, 1995
; Rothen-Rutishauser et al., 1998
). Microtubule minus-end-directed motors like cytoplasmic dynein (Fath et al., 1997
; Lafont et al., 1994
; Wang et al., 2003
) or KIFC3 (Noda et al., 2001
) transport cargo such as membrane vesicles towards the apical surface, whereas conventional kinesin catalyses transport to the basolateral surface (Lafont et al., 1994
).
Since HSV-1 virions and capsids are too large to be transported efficiently in the cytoplasm by diffusion (Sodeik, 2000), this raises the question of how incoming HSV-1 capsids reach the nuclear pores after inoculation of highly polarized epithelial cells from either the apical or basolateral plasma membrane. If HSV-1 fused with the apical plasma membrane and the cytosolic capsids bound to dynein and dynactin, they would be transported back to the apical surface rather than to the nucleus. Therefore, entry of HSV-1 by fusion with the apical plasma membrane would require a plus-end-directed microtubule motor for capsid transport to the nucleus. Alternatively, HSV-1 could enter by endocytosis and intact virions could use endocytic membrane traffic for transport from the apical surface to the nucleus. This scenario requires that viral fusion does not occur until the endosomes have moved close to the nuclear pores. Moreover, on entry from the basolateral surface, the minus-end-directed microtubule motor dynein would transport capsids towards the apical compartment if the capsids did not detach from the microtubules in time to switch over to the nucleus, which is located approximately in the basolateral half of the cells.
To address these questions, we set up polarized epithelial cell culture systems using MDCK and Caco-2 cells. In contrast to previous reports, our data showed that highly polarized epithelial cells were not susceptible to apical infection. However, HSV-1 infected these cells if added from the basolateral surface or if a depletion of extracellular Ca2+ had weakened the strength of cellcell contacts. Basolateral infection and apical infection after a Ca2+ switch required an intact microtubule network. This system can now be used to identify the microtubule motors that HSV-1 uses in polarized epithelial cells.
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METHODS |
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Antibodies.
To monitor age-related changes of the epithelial cells, we used an affinity purified rabbit antiserum to occludin (Zymed Laboratories), the mouse monoclonal antibody (mAb) 34 to E-cadherin (BD Biosciences Clontech), the mouse mAb DM1A (Sigma-Aldrich) to label tubulin, and mouse mAbs to gp114 as marker for the apical and to gp 58 for the basolateral surface (Balcarova-Stander et al., 1984). The major receptor for HSV-1, nectin-1, was detected with the mouse mAb CK6 (Krummenacher et al., 2000
), the immediate-early HSV-1 protein ICP4 with the mouse monoclonal 58S (Showalter et al., 1981
) and the late HSV-1 protein gB with a rabbit polyclonal serum R68 (Eisenberg et al., 1987
). Filamentous actin was visualized using FITCphalloidin (Sigma-Aldrich).
Virological techniques.
Wild-type HSV-1 strain F (ATCC VR-733), wild-type HSV-1 strain 17+ (from John Subaq-Sharpe) and the -galactosidase-expressing strain [KOS]tk12 (Warner et al., 1998
), which expresses the bacterial lacZ gene encoding the enzyme
-galactosidase under the control of the immediate-early ICP4 promoter of HSV-1, were amplified in BHK cells, purified and titrated in Vero cells as described (Döhner et al., 2002
; Sodeik et al., 1997
).
To depolymerize microtubules, the cells were treated with nocodazole (Sigma-Aldrich,) for 1 h prior to virus infection, then kept in nocodazole for the duration of the experiment. To deplete extracellular Ca2+, cells were incubated with 10 mM EGTA at 37 °C for 30 or 45 min prior to the addition of virus.
Cells were infected with virus diluted in CO2-independent culture medium (Gibco Life Technologies) supplemented with 0·2 % (w/v) cell-culture grade BSA (Sigma-Aldrich). The virus suspension was added at 0·5 ml per well or filter to the cells for 1 h on ice for cells grown on plastic and at room temperature for cells grown on filters, since the latter could not be maintained well at 4 °C. After inoculation, the virus was removed and the cell dishes were incubated in a water bath at 37 °C in CO2-independent culture medium supplemented with 10 % foetal calf serum for another 4·5 h.
Galactosidase assay.
Immediate-early viral gene expression was quantified using the mutant HSV1[KOS]tk12 as described (Mabit et al., 2002). The amount of
-galactosidase was determined after lysis in 0·5 % (v/v) TX-100/PBS with 1 mg BSA ml-1 and protease inhibitors using O-nitrophenyl
-D-galactosylpyranoside as a substrate. The lysate was incubated with the substrate for about 2 h at room temperature and the enzymic activity (A420) was measured using a plate reader (Spectra Count Microplate Photometer; Packard Instruments Company). The cell density was estimated with a parallel set of plates or filters by staining fixed cells with 0·25 mg crystal violet ml-1 in 5 % (v/v) ethanol for 5 min. After drying, bound crystal violet was dissolved in 100 % ethanol and the A590 was read.
Microscopy.
Cells grown on cover slips were fixed and labelled essentially as described previously (Döhner et al., 2002; Sodeik et al., 1997
). Cells grown on membrane filters were fixed with 3 % (w/v) paraformaldehyde in PBS for 30 min followed by treatment with 50 mM NH4Cl in PBS for 20 min and 0·1 % Triton X-100 in PBS for 10 min. Non-specific antibody binding was quenched using 0·2 % (v/v) cold-water-fish skin gelatin (Sigma-Aldrich) and 0·5 % (w/v) BSA in PBS for 3060 min prior to labelling with the antibodies, diluted in the same buffer, for 3060 min at room temperature. Where indicated, nuclei were labelled with 20 µg propidium iodide ml-1 after treating the permeabilized cells with 1 mg RNase A ml-1 (Sigma-Aldrich) in PBS for 10 min at room temperature (Reinsch et al., 1998
). Affinity-purified secondary antibodies were purchased from Dianova. The cells were mounted in Moviol containing 50100 mg 1,4-diazabicyclo(2,2,2)octane (DABCO) ml-1 or 25 mg N-propylgallate ml-1 and examined with a fluorescence microscope equipped for laser-scanning confocal light microscopy (DM IRB/E; Leica). Optical sections were recorded using a 100x oil immersion objective with a numerical aperture of 1·4 at a resolution of 512x512 pixels. Digitalized images were further processed using Adobe Photoshop version 4.0.
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RESULTS |
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These experiments showed that the peak in TER resistance at day 2 corresponded with the formation of a confluent monolayer of epithelial cells of which some already showed a polarized distribution of integral plasma membrane proteins. Around day 45, the TER was stabilized and the separation, as well as polarization, of apical and basolateral plasma membrane domains had been completed. These observed changes of the TER over the culture period agreed well with results from Rothen-Rutishauser et al. (1998).
HSV-1 infection of unpolarized MDCK cells
Several laboratories have reported the infection of polarized epithelial cells with HSV-1 after virus challenge from both the basolateral and the apical plasma membrane (Griffiths et al., 1998; Sears et al., 1991
). When we used a viral mutant expressing the lacZ gene under the control of an immediate-early HSV-1 promoter (Warner et al., 1998
) to infect our clone of MDCK cells cultured on plastic dishes, there was a dose-dependent increase in the synthesis of galactosidase (Fig. 2
A). If, however, the cells were infected in the presence of nocodazole, which reversibly depolymerizes microtubules, the synthesis of
-galactosidase decreased in a concentration-dependent manner (Fig. 2B
). Infecting cells grown on cover slips with wild-type HSV-1 for 4 h resulted in a prominent nuclear labelling for the immediate-early protein ICP4. Interestingly, the cells were not homogeneously infected; HSV-1 showed a clear preference for isolated cells. As also reported by Schelhaas et al. (2003)
, the peripheral cells of an MDCK islet contained more ICP4 when compared with cells completely surrounded by other cells in a confluent region (Fig. 2C
).
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Basolateral infection of polarized epithelial cells requires microtubules
We next wanted to analyse the infection of fully polarized MDCK cells via the apical plasma membrane. However, we could detect only very low levels of -galactosidase synthesis when cells cultured on filters for 7 days were infected with HSV-1 over a wide range of multiplicities (not shown). Confocal laser-scanning microscopy analysis revealed a few isolated cells labelled for ICP4, but overall there were few signs of an HSV-1 infection (Fig. 4
A). As previously reported, we were also unable to infect MDCK cells grown on 0·4 µm pore filters from the basal chamber with herpesvirus (Hemmings & Guilbert, 2002
; Topp et al., 1997
). If, however, MDCK cells were grown for 8 days on filters with a larger pore diameter of 3 µm, which allowed passage of HSV-1 virions, and infected from the basolateral chamber, the cells were clearly permissive for HSV-1 infection and many were labelled by antibodies against ICP4, an immediate-early HSV-1 protein, and gB, a late structural protein (Fig. 4B
). Infection via the basolateral plasma membrane was also inhibited by nocodazole in a dose-dependent manner (Fig. 4C
). Thus, MDCK cells differentiated on filters were susceptible to infection, but only if HSV-1 had access to the basolateral surface of the cells and not if the virus was added to the apical chamber.
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Apical infection of fully polarized human Caco-2 cells is facilitated by depleting extracellular Ca2+
The lack of susceptibility of the canine MDCK cells once they are fully polarized to apical infection with the human pathogen HSV-1 might be due to a host species barrier. Therefore, we performed additional experiments using the human cell line Caco-2, which also differentiates and polarizes if grown on porous filters (Rousset, 1986). Initial experiments using cells grown on plastic showed that our Caco-2 cell clone was fully permissive to HSV-1 infection and synthesized large amounts of galactosidase (Fig. 6
A). We next monitored cell differentiation during culture on porous filters. Caco-2 cells developed differently compared with the MDCK cells (cf. Fig. 1A
). The TER of the Caco-2 cells was very low at day 1, but steadily rose to reach 300 Ohm·cm2 by day 13 (Fig. 6B
). The development of TER was similar to the results reported by Griffiths et al. (1998)
for Caco-2 cells, but the absolute overall resistance was lower in our experiments. Infection of this Caco-2 clone with HSV-1 from the apical chamber yielded similar results to those obtained with the MDCK cells. The Caco-2 cells were susceptible to HSV-1 infection after culture for 6 days but not for 12 days. If, however, the cells were treated with EGTA to deplete Ca2+, they became susceptible to HSV-1 infection from the apical surface (Fig. 6C
).
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DISCUSSION |
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However, our data showed that fully polarized MDCK cells could not be infected with HSV-1 added to the apical culture chamber. We first determined that our clone of MDCK cells was fully susceptible to HSV-1 by showing that unpolarized cells were easily infected by HSV-1, irrespective of whether they were grown on a plastic surface, on glass cover slips or on filters for 2 days. We then used two assays to assess the differentiation state of the filter cultures: the development of the TER and the subcellular localization of integral plasma membrane proteins known to develop a polarized distribution. According to these criteria, we focused our experiments on MDCK cells that had either been cultured on filters for 2 days and had not yet developed two separated plasma membrane domains, or had been cultured for longer than 5 days, which we classified as maximally polarized. The latter is most likely the cause of major differences compared with previously published reports, which used younger epithelial cultures on filters for their experiments.
As reported previously for the epithelial Vero and PtK2 cell lines (Döhner et al., 2002; Mabit et al., 2002
; Sodeik et al., 1997
), efficient infection of unpolarized as well as highly polarized MDCK cells from the basolateral or apical plasma membrane after Ca2+ depletion required an intact microtubule network. Nocodazole, a reversible inhibitor of microtubule assembly (Jordan & Wilson, 1999
), at a concentration of 33 µM was sufficient to completely depolymerize the MDCK microtubule network under all culture conditions used (data not shown; but see also Grindstaff et al., 1998
; Lafont et al., 1994
). There are numerous reports that nocodazole does not have non-specific effects on protein synthesis in polarized epithelial cells (see, for example, Eilers et al., 1989
; Grindstaff et al., 1998
). Moreover, nocodazole does not affect either HSV-1 binding to cells or internalization, but reduces cytosolic capsid transport to the nucleus (Sodeik et al., 1997
). In the MDCK cells analysed in this study, nocodazole most likely inhibited either the cytoplasmic transport of endocytosed HSV-1 or the cytosolic HSV-1 capsid transport. Both scenarios would reduce the number of viral genomes imported into the nucleus and, consequently, immediate-early gene transcription, as well as protein expression, which we quantified using a reporter virus expressing
-galactosidase.
Interestingly, HSV-1 infected single MDCK cells or peripheral cells of a cell islet rather than confluent cells. In older cultures on filters, there were very few cells that showed any viral protein synthesis. However, this was not due to cell death since the cells were highly susceptible if the virus had access to the basolateral surface of cultures grown on large-pore filters or if the tight junctions and the adherens junctions had been opened experimentally by depleting extracellular Ca2+.
One might argue that the canine MDCK cell line is not the best model to study human viruses. However, we obtained similar results with the human Caco-2 cell line. Again, less polarized cells were more readily infected from the apical chamber than fully polarized cells. Moreover, infection experiments after pre-incubation with EGTA demonstrated that the older cultures were also fully susceptible to HSV-1 infection if the cellcell contacts had been opened.
As the cultures differentiate, two barriers for apical HSV-1 infection develop. One is that the major HSV-1 gD receptor, nectin-1, is increasingly sequestrated into adherens junctions. The second is the formation of tight junctions above the adherens junctions, which form such a tight seal between neighbouring cells that even small molecules such as sugars can hardly pass (Takai & Nakanishi, 2003). The second seems to be the major obstacle, since polarized cells were infected if the virus was able to access the cells from the basolateral chamber via large-pore filters. Moreover, heparan sulfate proteoglycans, important receptors for HSV-1 gC, are mainly localized in the basolateral plasma membrane (Caplan et al., 1987
), while the apical plasma membrane contains chondroitin sulfate proteoglycans (Kolset et al., 1999
), which cannot be used as efficiently by HSV-1 (Mardberg et al., 2002
).
Our experiments are in agreement with and extend the studies of Yoon & Spear (2002) and Schelhaas et al. (2003)
. Even MDCK cells grown to confluency on glass or plastic are rather resistant to apical HSV-1 infection until the monolayer has been wounded or the cellcell contacts weakened by cytochalasin treatment (Schelhaas et al., 2003
). Moreover confluent MDCK cells, stably transfected with human nectin-1 and grown on plastic or glass, bind more soluble gD and are more efficiently infected with HSV-1 when the adherens junctions have been disrupted by Ca2+ depletion (Yoon & Spear, 2002
). These results and ours with highly polarized MDCK cells expressing endogenous nectin-1 suggest that, under steady-state conditions, most of the nectin-1 molecules are engaged in homotypic interactions. More nectin-1 receptors become available for virus binding if the depletion of extracellular Ca2+ releases these interactions. Thus, the entry of HSV-1 into polarized cells and infection require access to microtubules and to receptors present in the adherens junction. Our data suggest that an intact, fully polarized epithelium that has established tight junctions is not susceptible to apical HSV-1 infection. This supports the hypothesis proposed by Spear (2002)
that a primary infection might only occur via small wounds or via undifferentiated and thus unpolarized cells present in the mucosa.
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ACKNOWLEDGEMENTS |
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Received 25 July 2003;
accepted 2 December 2003.