Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells

Mario Schelhaas1, Matthias Jansen1, Ingo Haase2 and Dagmar Knebel-Mörsdorf1,3

1 Max Planck Institute for Neurological Research, University of Cologne, Gleuelerstrasse 50, D-50931 Cologne, Germany
2 Department of Dermatology, University of Cologne, Gleuelerstrasse 50, D-50931 Cologne, Germany
3 Department of Neurology, University of Cologne, Gleuelerstrasse 50, D-50931 Cologne, Germany

Correspondence
Dagmar Knebel-Mörsdorf
D.Moersdorf{at}pet.mpin-koeln.mpg.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Herpes simplex virus type 1 (HSV-1) enters its host via epithelia and spreads to neuronal cells where latency is established. Hence, the in vivo route of infection relies on penetration and subsequent passage of HSV-1 through highly polarized cells. Infection studies were performed in both polarized MDCKII cells and primary human keratinocytes to gain insight into the pathway of virus entry into individual epithelial cells. Early viral gene expression was barely detectable in confluent MDCKII cells, even at high m.o.i. However, after wounding the cell layer, infected cells were observed next to the wound, where basolateral membranes were accessible. In subconfluent monolayers, MDCKII cells are organized in islets. After infection, viral capsids and early viral gene expression were detectable in peripheral cells of islets, supporting virus penetration via basolateral membranes. Further infection studies were performed in human keratinocytes, which represent the primary target cells for HSV-1 infection in vivo. In primary keratinocytes grown as monolayer cultures and wounded prior to infection, HSV-1 infection led to early viral gene expression predominantly in cells next to the wound. When stratifying cultures of primary human keratinocytes were infected, early viral gene expression was localized to peripheral cells of basal keratinocytes. Finally, infection of epithelial tissue such as human foreskin epithelia demonstrated HSV-1 entry exclusively via basal cell layers. Staining of the potential coreceptor nectin-1/HveC revealed no correlation of receptor localization and virus entry sites in keratinocytes. These results provide first evidence for a virus entry mechanism that relies on the accessibility to basal surfaces of epithelial tissue and to basolateral membranes, both in MDCKII and primary keratinocytes.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Herpes simplex viruses (HSV) enter their host via mucosal epithelia, skin or cornea. While humans are the only natural host, HSV can infect a wide range of mammalian species under laboratory conditions, indicating a broad host range. Primary infection in the epidermis is followed by penetration of the nervous system and establishment of latent infection in neurons. Upon reactivation, HSV is transported back to the epithelium to reinfect epithelial cells. Thus, the infection route in vivo implies that viruses gain access to highly polarized cells.

The cellular entry process of HSV involves multiple steps and relies on the interaction of several viral glycoproteins with various cell surface receptors. The initial step involves attachment of the virus particles and is mediated by the HSV envelope glycoprotein C (gC) and/or gB with cell surface heparan sulphate proteoglycans (WuDunn & Spear, 1989; Herold et al., 1994). This initial contact facilitates subsequent binding to a coreceptor, which is required for entry into the cell. The viral envelope protein gD appears to serve as virus ligand for all HSV coreceptors identified so far. gD coreceptor binding is followed by fusion of the viral envelope and the plasma membrane, which is triggered in concert with further viral envelope glycoproteins, such as gB, gH and gL (reviewed by Spear, 1993). At least three classes of cell surface molecules can act independently as coreceptors. One is a member of the TNF receptor family, four are related to members of the immunoglobulin superfamily and another belongs to the protein family of sulphotransferases (reviewed by Campadelli-Fiume et al., 2000; Spear et al., 2000). To date, the most intensively studied gD coreceptor is nectin-1 (named HveC) (Cocchi et al., 1998; Geraghty et al., 1998), an immunoglobulin-like intercellular adhesion molecule (reviewed by Takai & Nakanishi, 2002).

While our knowledge of the HSV entry process is based upon infection studies in nonpolarized cells, the entry mechanism in polarized epithelia remains elusive. Polarized epithelial cells exhibit a polar distribution of proteins and lipids in the plasma membrane creating two distinct surface domains: the apical surface, which faces the external environment, and the basolateral surface, which contacts the underlying and neighbouring cells (Simons & Fuller, 1985). It has become apparent that both the entry and the release of many viruses from epithelial cells are highly polarized, occurring selectively at either the apical or the basolateral sites. Directional virus entry and release may have important implications in pathogenesis (reviewed by Blau & Compans, 1996). HSV entry has been observed from either cell surface of polarized epithelial cells, such as MDCK cells (Madin–Darby canine kidney cells) (Sears et al., 1991; Tran et al., 2000), or with a preference via the basolateral surfaces of MDCKII cells (Topp et al., 1997). In addition, enhanced HSV infection is evident when viruses are applied to MDCK cells after disruption of cell–cell contacts by calcium depletion as compared to poor infection of confluent MDCK cells (Hayashi, 1995; Yoon & Spear, 2002). The authors conclude that dissociation of cell contacts releases the cell adhesion molecule nectin-1 to serve more efficiently as a virus receptor (Yoon & Spear, 2002).

We aim to understand how HSV-1 enters epithelia in vivo. The focus of our studies is the cellular mechanism that is used by virus particles to enter their host cell efficiently in a stratified epithelium. It is a general assumption that viruses pass through microlesions to infect mucosa, skin or cornea. Yet the underlying mechanism of how viruses gain access to specific receptors and enter individual cells in the epithelium has still to be elucidated. One precondition for the understanding of the molecular determinants is the identification of the initial virus entry site. Here, we report on the polar entry of HSV-1 into the epithelial MDCKII cell line, primary human keratinocytes and human foreskin epithelia. HSV-1 entry into individual cells was visualized by staining infected cells with an antibody directed against the HSV-1 early regulatory protein ICPO. Staining of ICPO indicates viral gene expression immediately after infection and exhibits a distinct localization pattern in the cell nucleus, thus allowing the identification of infected cells prior to virus replication (Maul et al., 1993; reviewed by Everett, 2000). Based on capsid and ICPO stainings, our results strengthen a cellular entry mechanism via basolateral membranes of epithelial cells and provide first insights into the entry process in primary keratinocytes, which form the primary target epithelium for HSV-1 infection in vivo.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
MDCKII cells (Louvard, 1980; Hansson et al., 1986) and BHK cells (ATCC) were maintained in DMEM (Invitrogen) containing 10 % FCS (Boehringer), penicillin (100 IU ml-1) and streptomycin (100 µg ml-1). Epidermal sheets were prepared from normal human foreskins obtained from circumcisions. Fat and most of the connective tissue were removed using curved scissors. Skin pieces of 6 mm diameter were cut out and incubated for 15–18 h on ice with 10 mg dispase II ml-1 (Boehringer) in DMEM containing penicillin/streptomycin. Using forceps, the epidermis was gently removed from the underlying dermis as an intact sheet and used immediately for infection studies. Primary human foreskin keratinocytes were prepared and cultured on feeder layers as described (Watt, 1998). Briefly, foreskin epidermis was incubated with 0·25 % trypsin, the obtained suspension was filtered through gauze filters, collected, resuspended and seeded into culture dishes containing NIH-3T3 feeders, strain J2. In order to enable stratification, primary human keratinocytes were maintained in keratinocyte culture medium (Ham's F12/DMEM 1 : 3, Invitrogen) containing 1·8 mM calcium ions, 10 % FCS, penicillin (100 IU ml-1), streptomycin (100 µg ml-1), adenine (1·8x10-4 M), glutamine (2 mM), hydrocortisone (0·5 µg ml-1), epidermal growth factor (10 ng ml-1), cholera enterotoxin (10-10 M), insulin (5 µg ml-1) and vitamin C (50 µg ml-1) in the presence of mitomycin C-treated feeder cells. Unstratified monolayer cultures of keratinocytes were obtained by decreasing the calcium concentration of the culture medium to 50 µM.

MDCKII and keratinocyte monolayers were wounded 1 h prior to infection by scratching with a 27-gauge needle and a glass stick, respectively.

Subconfluent MDCKII cells were treated with cytochalasin D (CD) (Sigma) (stock solution 10 mM in ethanol) at 30 min prior to infection. Cells were then incubated in the presence of CD until the virus inoculum was removed at 1 h post-infection (p.i.).

Viruses.
Infection studies were performed with HSV-1 strain 17 (McGeoch et al., 1988), a wild-type clinical isolate, at low or high m.o.i., as indicated. Time 0 was defined as the time when the virus inoculum was added to the cells. Virus inoculum was prepared by infecting monolayers of confluent BHK cells at a multiplicity of 0·003 p.f.u. per cell at 31 °C. At 24 h p.i., the inoculum was replaced by culture medium. For infection studies, only purified virus particles derived from the supernatant of infected BHK cells were used. Virus was concentrated from the culture medium at about 120 h p.i. by centrifugation for 90 min at 12 500 r.p.m. The virus suspension was then loaded onto a linear 5–15 % Ficoll400 (Sigma) gradient and centrifuged for 2 h at 12 000 r.p.m. Virus from the virion band was pelleted for 90 min at 21 000 r.p.m. and resuspended in DMEM.

Immunocytochemistry and antibodies.
MDCKII cells were grown on glass coverslips, whereas primary keratinocytes were grown on Lab-Tek Permanox chamber slides (Nunc). Cells were fixed in 2 % paraformaldehyde for 15 min at room temperature, washed three times with PBS and permeabilized by incubation with 0·1 % Triton X-100 for 15 min. Subsequently, cells were blocked with 3 % BSA in PBS for 30 min and then incubated for 1 h with primary antibodies using a 1 : 500 dilution for rabbit anti-ICP0 antiserum (r191) (Parkinson & Everett, 2000), a 1 : 2000 dilution for mAb 11060 (mouse anti-ICPO) (Everett et al., 1993), a 1 : 100 dilution for mAb DM165 (mouse anti-HSV capsid) (McClelland et al., 2002), a 1 : 250 dilution for mAb 1520 (rat anti-ZO-1, IgG) (Chemicon), a 1 : 500 dilution for mAb hecD (mouse anti-E-cadherin) (Zymed) and a 1 : 250 dilution for mAb CK41 (mouse anti-nectin-1) (Krummenacher et al., 2000). All antibodies were diluted in PBS containing 3 % BSA.

Primary human foreskin epithelia were fixed in 2 % paraformaldehyde for 90 min at room temperature, washed with PBS and infiltrated with Jung tissue freezing medium (Leica) overnight at 4 °C. Tissue was frozen in liquid nitrogen and 10 µm frozen cross-sections were cut using a Jung CM3000 (Leica) microtome. After two washes with PBS, tissue sections were blocked with 3 % BSA for 60 min and then incubated for 90 min with mouse mAb 11060 (anti-ICP0), at a 1 : 2000 dilution, or mouse mAb hecD (anti-E-cadherin), at a 1 : 500 dilution, both in PBS containing 3 % BSA.

Primary antibodies were visualized with fluorochrome-conjugated anti-rabbit, anti-mouse or anti-rat IgG. Staining of F-actin was achieved using TRITC- or FITC-conjugated phalloidin (Sigma). Specimens were mounted in Citifluor AF100 (Citifluor) and viewed under a Zeiss Axiovert 135 microscope with an Intas digital camera system. Cells were observed and images captured with either a 10x objective and a 0·25 numerical aperture (NA), a 20x objective and a 0·4 NA or a 100x objective and a 1·30 NA. Confocal imaging was performed under a Leica DM RE microscope linked to a Leica TCS-SP/2. Cells were observed and images captured with either a 40x objective and a 1·25 NA or a 63x objective and a 1·32 NA. Images were assembled using Adobe Photoshop, version 6.0.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Experimental design
For entry studies into polarized cells, we initially chose MDCKII cells, a subclone derived from the heterogeneous parental line MDCK, which is the most extensively studied polarized epithelial cell line (Louvard, 1980; Hansson et al., 1986). Confluent MDCKII cells represent a polarized epithelial monolayer. Prior to confluency, MDCKII cells grow in cell islets of various sizes. Since we performed infection studies in both confluent and subconfluent MDCKII cells, we determined cell polarity of subconfluent MDCKII cells by staining zonula occludens protein 1 (ZO-1), a molecular component of tight junctions. Tight junctions (or zonula occludens) are typically aligned at the apical side of lateral membranes in polarized epithelial cells (reviewed by Gonzalez-Mariscal et al., 2000). Confocal imaging indicated ZO-1 staining as belts that circumscribed the cells at the apical surfaces (Fig. 1B) and demonstrated their localization at the apical side of lateral membranes (Fig. 1C), which was consistent with the formation of tight junctions (Gottardi et al., 1996). In contrast, ZO-1 was barely detectable at the basolateral surfaces, only some staining was observed at the periphery of cell islets (Fig. 1A). Staining of epithelial cadherin (E-cadherin) visualized another type of epithelial cellular junctions (Aberle et al., 1996). The presence of E-cadherin throughout the lateral membranes indicated the polarized junctional alignment in MDCKII cells (data not shown). The formation of apical-basolateral surfaces in cell islets was visualized further by costaining the actin cytoskeleton, which demonstrated stress fibres at the basal side and apical microvilli (Fig. 1A, B). These results show that under subconfluent conditions, islets of MDCKII cells grown on glass surfaces exhibit characteristics of polarized epithelia.



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Fig. 1. Characteristics of subconfluent MDCKII cells. Subconfluent MDCKII cells were fixed in 2 % paraformaldehyde at 16 h after seeding. The actin cytoskeleton was visualized with TRITC-conjugated phalloidin (red). ZO-1 staining was performed with rat anti-ZO-1 antibodies and FITC-conjugated anti-rat IgG (Sigma) (green). Confocal images of the basal (A) and apical planes are shown (B). The XZ section is also shown (C). The merges of the staining are shown.

 
Throughout this study, we used the clinical isolate HSV-1 strain 17 as wild-type strain (McGeoch et al., 1988). Since cellular contamination of the virus inoculum might interfere with the initial steps of virus entry, infection studies were performed with purified virus stocks of high titre.

HSV-1 infection of confluent and subconfluent MDCKII cells
Our interest concentrates on the cellular virus entry site; hence, cells were fixed at 2 h p.i. Infected cells were visualized by staining with an antibody directed against the HSV-1 protein ICPO to detect immediate early viral gene expression. Counterstaining of the actin cytoskeleton served as marker for the individual cell shape and the size of cell islets.

When infection studies were performed in fully confluent MDCKII cells, we observed only very few infected cells, even at high m.o.i. (Fig. 2A, B). In contrast, after infection of subconfluent MDCKII cells, ICPO staining was detectable preferentially at the rim of cell islets (Fig. 2C, D). Staining of ICPO in peripheral cells correlated with the staining pattern of viral capsids. At 2 h p.i., the anti-capsid antibodies stained virus particles predominantly in the peripheral cells of cell islets (Fig. 2E, F). Thus, capsid and ICPO staining suggest that penetration of HSV particles is restricted to the peripheral cells of cell islets.



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Fig. 2. HSV-1 infection of confluent versus subconfluent MDCKII cells. Confluent MDCKII cells were infected at 3 days after seeding with (A) 10 or (B) 100 p.f.u. per cell. Subconfluent cells were infected 16 h after seeding with (C, D, G) 2–5 or (E, F, H) 100 p.f.u. per cell. Infection with high multiplicity was performed to visualize a sufficient number of capsids (E, F) or to demonstrate infected cells in more central parts of cell islets (H). After fixation at 2 h p.i., cells were costained with FITC-conjugated phalloidin (green) and with either mouse anti-ICPO (A–D) or mouse anti-capsid (E, F) visualized by Cy3-conjugated anti-mouse IgG (Jackson) (red). Cells were costained with rabbit anti-ICPO antibodies and rat anti-ZO-1 visualized with Cy3-conjugated anti-rabbit IgG (red) and Alexa Fluor 488 anti-rat IgG (Molecular Probes) (red), respectively (G, H). Double-label immunofluorescence analyses are shown as single staining (E) or as overlays (A–D, F). The merge of confocal projections is also presented (G, H). Infected cells are indicated by arrowheads (B).

 
Only when high m.o.i. of virus (100 p.f.u. per cell) were applied did we occasionally observe infected cells present in more central parts of the islets (Fig. 2H). In these islets, ZO-1 was localized tightly at the apical sides of lateral membranes, which was similar to the staining of islets with only peripheral infection and indicated polarization of the cells within the islets (Fig. 2G, H). Interestingly, we observed a variation in the ICPO staining pattern at 2 h p.i. It is known that ICPO localizes in the nucleus at the onset of infection, giving a punctate staining pattern in a diffuse background (Maul et al., 1993). As infection progressed, we observed that the diffuse nuclear staining pattern became dominant and was followed by the relocalization to the cytoplasm (data not shown). At 2 h p.i., infected cells within islets showed nuclear staining of ICPO, while cytoplasmic staining was detectable in the peripheral cells (Fig. 2H). These results provide evidence that infection occurs first in peripheral cells and is then followed by a delayed infection of cells next to the periphery.

Treatment of cells with CD leads to inhibition of F-actin polymerization and results in the disruption of cell–cell contacts. When increasing amounts of CD were added to MDCKII cells prior to infection, staining of ICPO demonstrated uniformly infected cells throughout the islets (Fig. 3A). These results indicate that the virus particles gain access to all cells once they loose their cell–cell contacts and become depolarized.



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Fig. 3. HSV-1 infection of CD-treated or wounded MDCKII cells. (A) Subconfluent MDCKII cells were untreated (panel a) or treated with 250 nM (panel b) and 1 µM of CD (panel c) followed by infection with HSV-1 (5 p.f.u. per cell). (B) Confluent MDCKII cells were wounded prior to infection and staining patterns are shown with different magnifications, as indicated. At 2 h p.i. cells were fixed and stained with FITC-conjugated phalloidin (green) and with mouse anti-ICPO and Cy3-conjugated anti-mouse IgG (Jackson) (red). The overlays of the double-staining immunofluorescence analyses are shown.

 
ICPO staining reflects only early viral gene expression. Hence, we performed time-course experiments to demonstrate productive HSV-1 infection in subconfluent MDCKII cells, which resulted in a significant cytopathic effect (Fig. 4A, B). Conclusively, we designated cells with early viral gene expression as infected cells. As compared to HSV-1 growth curves in Vero cells, the peak of virus production in MDCKII cells was delayed by about 24 h (data not shown). After infection of fully confluent MDCKII cells, nearly no cytopathic effect was detectable, which was in line with the observation of rare ICPO staining (Fig. 4C, D).



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Fig. 4. Cytopathic effect after infection of subconfluent or confluent MDCKII cells. Subconfluent MDCKII cells were mock-infected (A) or infected 16 h after seeding with 5 p.f.u. per cell (B). Confluent MDCKII cells were mock-infected (C) or infected at 3 days after seeding with 5 p.f.u. per cell (D). Cytopathic effect is shown 24 h p.i. by phase contrast. Magnification, x20.

 
In summary, our results demonstrate that the efficacy of infection by HSV-1 is extremely low in MDCKII cells forming a confluent, polarized sheath but increases dramatically upon disruption of polarity and cell–cell contacts by CD treatment. We conclude that efficient infection can occur only if basolateral membranes become accessible for HSV-1.

HSV-1 infection of wounded MDCKII cell sheets
To gain further insight into how the status of the MDCKII cells may determine virus entry, we performed infection studies in polarized MDCKII cell layers that were wounded prior to infection. At 2 h p.i., ICPO staining was only detectable in cells next to the wound (Fig. 3B, panels a and b). Interestingly, some cells exhibited prominent cytoplasmic staining of ICPO, which indicated higher susceptibility to virus infection than neighbouring cells (Fig. 3B, panels b and c). These results further support a virus entry mechanism via basolateral membranes of MDCKII cells.

HSV-1 infection of primary human keratinocytes
Since skin or mucosa represent the primary target epithelium for HSV-1 infection in vivo, we performed infection studies in human keratinocytes at various cell densities. Preparations of primary keratinocytes were grown under low calcium conditions to maintain a monolayer of relatively undifferentiated basal cells. We observed that the variations in the susceptibility of keratinocytes to HSV-1 infection was related to variations in the degree of cell–cell contact formation, depending on cell density in the culture dish. Costaining of F-actin and ICPO showed that monolayers with low density and poor cell–cell contact formation exhibited a high percentage of keratinocytes staining positive for ICPO, whereas confluent monolayers that clearly formed cell–cell contacts showed little or no ICPO staining (Fig. 5, compare A and C to B). When dense monolayers with a high degree of cell contacts were wounded prior to infection, ICPO staining was observed readily in nearly all cells close to the wound (Fig. 5D).



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Fig. 5. HSV-1 infection of primary human keratinocytes. Different preparations of undifferentiated basal keratinocytes were grown as monolayers (A–C) and were wounded prior to infection (D). Monolayer (A–D) and stratified cultures of primary keratinocytes (E, F) were infected with 50 p.f.u. per cell. At 2 h p.i. cells were fixed and stained with either TRITC-conjugated phalloidin (red) and mouse anti-ICPO visualized by Alexa Fluor 488 anti-mouse IgG (Molecular Probes) (green) (A, B, E, F) or with FITC-conjugated phalloidin (green) and with mouse anti-ICPO visualized by Cy3-conjugated anti-mouse IgG (red) (C, D). The merge of confocal projections (A, B), the overlay of immunofluorescence studies (C, D) and the basal (E) and apical (F) views of confocal sections are shown. (A, B) Different areas of the same monolayer culture. Poor cell–cell contacts (A) versus formed cell–cell contacts (B) are indicated by arrowheads.

 
We next examined HSV-1 infection in a three-dimensional cell culture system. Primary human keratinocytes were allowed to stratify, which resulted in a partially differentiated epithelium. After adding HSV-1 to the cell culture medium, cultures were stained with anti-ICPO antibodies at 2 h p.i. Interestingly, early viral gene expression was only detectable in peripheral cells of basal keratinocytes, even at high m.o.i. (~50 p.f.u. per cell) (Fig. 5E, F). Hence, we conclude that HSV-1 enters the stratified cell culture via the basal undifferentiated keratinocytes.

Nectin-1/HveC localization in primary human keratinocytes
The finding of polar entry in keratinocytes leads us to the intriguing question of whether the preferred polar entry site of HSV-1 correlates with the sorted distribution of a virus receptor. One putative candidate is the adhesion molecule nectin-1/HveC, which is broadly expressed in human cell lines (reviewed by Takai & Nakanishi, 2002). When primary keratinocytes grown as monolayers were investigated for the presence of nectin-1/HveC, we observed staining at cell–cell contacts, as visualized by costaining the actin cytoskeleton (Fig. 6A, B). Nectin-1/HveC localization at the plasma membrane was not restricted to lateral membranes but was also detectable at apical surfaces (Fig. 6C, D). In addition, some cells showed strong nectin-1/HveC staining in the cytoplasm (Fig. 6A, B). Interestingly, in wounded keratinocyte monolayers, nearly no staining was detectable in most cells next to the wound (Fig. 6E, F, arrow), yet some cells at the wound indicated cytoplasmic nectin-1/HveC staining (Fig. 6E, F, asterisk). The presence of nectin-1/HveC at intercellular junctions, however, was limited to cells in dense parts of the monolayer (Fig. 6E, F, arrowhead). Analysis of stratified keratinocyte cultures revealed prominent nectin-1/HveC staining at cell–cell contacts dominantly in the apical cell layers of differentiated keratinocytes (Fig. 6G, H). In contrast, nectin-1/HveC was only occasionally detectable in basal keratinocytes (data not shown). Conclusively, the staining pattern of nectin-1/HveC did not correlate with cells of preferred HSV entry.



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Fig. 6. Nectin-1/HveC localization in primary human keratinocytes. Basal keratinocytes were grown as monolayers (A–D) and were wounded 1 h prior to fixation (E, F). Monolayer (A–F) and stratified cultures of primary keratinocytes (G, H) were fixed and stained with either FITC-conjugated phalloidin (green) and with mouse anti-nectin-1 visualized by Cy3-conjugated anti-mouse IgG (red) (A–F) or with TRITC-conjugated phalloidin (red) and mouse anti-nectin-1 visualized by Alexa Fluor 488 anti-mouse IgG (green) (G, H). Confocal projections (A, E), the merge of confocal projections (B, F) and confocal images as XZ sections (C, D) and in the apical plane (G, H) are shown.

 
HSV-1 infection of human foreskin epithelia
We performed infection studies in human foreskin epithelium to mimic the infection route in vivo. The presence of cell contacts was confirmed by staining with anti-E-cadherin antibodies. As expected, the staining of cell–cell borders indicated the presence of E-cadherin at intercellular junctions throughout the cell layers up to the stratum granulosum (Fig. 7B).



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Fig. 7. HSV-1 infection of human foreskin epithelia. Foreskin epidermis was infected by floating the sheets with the basal surface (A, C) or apical surface (D–F) on keratinocyte culture medium. Virus was added to the medium at a calculated multiplicity of about 10 p.f.u. per cell (A) or of about 100 p.f.u. per cell (C–F). At 6 h p.i., tissue was fixed and cut, followed by staining of the tissue sections with TRITC-conjugated phalloidin (red) and with either mouse anti-ICPO (A, C–F) or mouse anti-E-cadherin (B) and Alexa Fluor 488 anti-mouse IgG (green). Tissue samples are shown with the apical surfaces on the top and the basal layers on the bottom. In all cases, unspecific staining of the cornified layer on top of the tissue samples was observed (green). The merge of confocal projections is shown.

 
Viruses were applied to the medium in which the epidermis of foreskin was maintained and epidermal sheets were fixed at 6 h p.i., followed by ICPO staining of tissue sections. To study entry into the polar tissue, epidermal sheets were allowed to float on the surface of virus-containing medium, thus having access to the basal epidermal layer only. Alternatively, sheets were submerged in virus-containing medium, giving HSV-1 access to all surfaces of the tissue. Using both infection procedures, virus entry was observed exclusively via the basal cell layers (Fig. 7A, C–F). With increasing virus titre, multiple cell layers extending to the spinous layer demonstrated ICPO staining (Fig. 7C–F). Only cells of the basal layer showed ICPO in the cytoplasm, while more peripheral cell layers contained cells with nuclear ICPO staining (Fig. 7F). Based on cellular ICPO localization, we conclude that virus infection occurs first in basal keratinocytes, whereas infection of more peripheral cell layers is most likely a consequence of basal cell infection or diffusion of virus into the intercellular epidermal spaces. Infection of suprabasal keratinocytes without infection of basal cells was never observed. From these experiments, we conclude that HSV-1 cannot infect human epidermis from the peripheral surface but infects keratinocytes with high efficiency once their basolateral membranes are exposed.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
By investigating HSV-1 entry into individual cells of confluent and subconfluent MDCKII cells, we provide evidence for a polar entry mechanism into epithelial cells. Even at high m.o.i., there was only occasional virus entry via apical surfaces of confluent MDCKII cells. However, once viruses had access to basolateral membranes either in subconfluent cells or in wounded monolayer cultures, efficient entry was observed. In addition, all cells were infected when cell–cell contacts were disrupted, which is in line with recent data demonstrating efficient infection of MDCK cells upon calcium depletion (Yoon & Spear, 2002).

Previous infection studies in MDCKII cells have been performed with cells grown on filters (Sears et al., 1991; Hayashi, 1995; Topp et al., 1997; Tran et al., 2000). In case of basolateral infection, one critical parameter of this procedure is that viruses have to pass the filter pores, which results in a loss of 99 % of the applied virus (Topp et al., 1997) or even more (data not shown). Therefore, our preferred model system was infection of subconfluent MDCKII cells grown on glass surfaces. In this model, cells were shown to exhibit characteristics of polarized cells. As a control, MDCKII cells grown on filters were infected via the apical surfaces, which demonstrated that even at high m.o.i. ICPO staining was barely observed (data not shown).

During infection studies of subconfluent cells, infected cells were observed only occasionally within MDCKII cell islets when high virus doses were applied. In contrast to the cytoplasmic staining of ICPO in peripheral cells, nuclear ICPO staining was present in cells within the islets. Since ICPO is present initially in the nucleus followed by relocalization to the cytoplasm, we conclude a delayed infection of cells that are parts of a polarized cell formation. One possible explanation is virus spreading from peripheral cells, which would require viral DNA replication. By adding the thymidine analogue BrdU to the cell culture medium prior to fixation of infected cells, viral DNA replication centres were visualized with anti-BrdU antibodies (de Bruyn Kops & Knipe, 1988). Costaining of replicating DNA and ICPO suggested a detectable onset of viral DNA replication in MDCKII islets at 2–3 h p.i. (data not shown). Hence, delayed infection within the islets could be the result of infection by newly synthesized virus particles. Alternatively, it is conceivable that virus functions in the peripheral cells facilitate virus access to cells within the islets by inducing alterations of cell–cell contacts.

Whether the entry mechanism observed in cell culture systems accounts for the virus entry into the epithelium of the natural host remains to be shown. In general, human keratinocytes become efficiently infected by HSV-1 (Huber et al., 2001). We infected primary cells at various stages to evaluate the polarity of HSV-1 entry into human keratinocytes. When virus was applied to monolayers of primary keratinocytes, we observed areas that were barely infected as well as areas of infected cells. Whereas in intact epidermis keratinocytes in vivo are tightly packed and have well-developed cell–cell contacts, cell density can show considerable variation in keratinocyte cultures that are low in calcium. We found that the efficacy of HSV-1 infection was inversely proportional to keratinocyte density and formation of cell–cell contacts in the culture dish. When dense monolayers were wounded, efficient virus entry was observed in keratinocytes that were close to the wound. This is comparable to our results obtained in wounded MDCKII monolayers. Conclusively, the accessibility of basolateral surfaces was a precondition for efficient virus entry in keratinocyte monolayers.

To validate our results obtained in cell cultures in a model that is closer to the situation in vivo, we prepared epidermal sheets from human foreskin. When these sheets were infected, we observed virus entry exclusively across the basal surface of the epidermis. In contrast, the apical surface of the epidermis formed a limiting barrier towards HSV-1 infection. With increasing virus dose, ICPO staining was also detectable in suprabasal cell layers. At the moment, we do not know whether this is due to virus spread from basal to suprabasal cells or whether HSV-1 at high concentrations is able to diffuse into the suprabasal epidermal layers. Taking into account the particle size of HSV-1 on one hand and the intact cell–cell contacts, as demonstrated by E-cadherin staining, on the other, we consider the latter possibility unlikely.

In the context of results obtained in stratifying cultures of immortalized or primary human keratinocytes in organotypic culture systems in vitro (Syrjänen et al., 1996; Visalli et al., 1997; Hukkanen et al., 1999), our findings elucidate further the mechanisms of HSV-1 action on epidermal cells by specifying the entry site of the virus into polarized epithelia. In several model systems, including epidermal explant cultures, we can show that cells that expose their basolateral membranes to the virus are infected with much higher efficacy than cells that expose apical surfaces only. In addition, our experiments with ex vivo cultures strongly suggest that in vivo HSV-1 does not penetrate through an intact epidermal barrier but requires the exposure of basal keratinocyte surfaces for successful infection. These results partly differ from those obtained in organotypic, stratifying cultures in vitro (Syrjänen et al., 1996; Hukkanen et al., 1999). It is well known, however, that such cultures do not represent the full barrier competence of epidermis in vivo (Nolte et al., 1993). In our view, explant cultures as used in this study are closer to the in vivo situation and are, therefore, more suitable to study HSV entry into epithelia.

We addressed whether the preferred polar entry site of HSV-1 correlates with the sorted distribution of the virus receptor nectin-1/HveC. In the keratinocyte cell line HaCaT, high levels of nectin-1/HveC have been observed (Huber et al., 2001). However, infection of HSV-1 is inefficiently blocked by anti-nectin antibodies (Huber et al., 2001). Our results indicated the presence of nectin-1/HveC at intercellular junctions of primary keratinocytes, which was most prominent in the differentiated cells of stratified cultures and in dense parts of monolayers. In contrast, nectin-1/HveC staining was virtually undetectable at the plasma membrane in cells close to a wound, and only observed occasionally in the basal cells of stratified cultures. This expression pattern of nectin, together with the inefficient blocking of HSV-1 infection by anti-nectin antibodies (Huber et al., 2001), raises doubts on a role of nectin as the exclusive HSV-1 receptor in keratinocytes. In addition, receptor accessibility may not be the only requirement for efficient virus entry. Based on our infection studies, we propose a model in which HSV-1 entry is enabled at sites of lesions in the epithelium, which implies access of the virus particles to basolateral surfaces of cells next to the lesion. These cells, in comparison to those in an intact epithelium, show numerous different characteristics, each of which could make them a preferred target for HSV-1 entry. Hence, it will be of considerable interest to investigate additional potential cellular mechanisms of virus entry into the epidermis in vivo.


   ACKNOWLEDGEMENTS
 
We thank Gill Elliot for advice and for kindly providing HSV-1 strain 17, Roger Everett for the antibodies against ICPO, Frazer Rixon for the antibodies against HSV capsid, Claude Krummenacher, Roselyn Eisenberg and Gary Cohen for the CK41 antibodies against nectin-1/HveC, Andrea Maisner and Gerrit van Meer for MDCKII cells, Ruth Pofahl for technical assistance and Beate Sodeik for discussion. Additional thanks go to Carien Niessen for critical and helpful comments on the manuscript.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 March 2003; accepted 19 May 2003.