Activation of Kaposi's Sarcoma-Associated Herpesvi
http://www.100md.com
病菌学杂志 2005年第21期
Departments of Laboratory Medicine
Otolaryngology, University of Washington, Seattle, Washington 98195
ABSTRACT
The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1 promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.
INTRODUCTION
Since its initial description over a decade ago (14), Kaposi's sarcoma-associated herpesvirus (KSHV) has been recognized as a significant viral pathogen, particularly for immunocompromised hosts and persons infected with human immunodeficiency virus type 1 (HIV-1). KSHV is the etiologic agent of Kaposi's sarcoma (KS), having been demonstrated in biopsy specimens of all forms of KS despite distinct differences in the geographic origin, age, and gender of affected persons (29). KSHV is also associated with multicentric Castleman's disease (80) and primary effusion lymphoma (11).
The precise modes of KSHV transmission are not clearly defined; however, epidemiological data suggest that both sexual and nonsexual routes are possible. The association between KSHV transmission and sexual activity has been largely defined in North American men who have sex with men (MSM), where risk factors include sex with a partner who has KS (66), a history of sexually transmitted infections (STIs) (55), and an increased number of sexual partners (55). Other risk factors, however, such as deep kissing with an HIV-positive partner (66) and orogenital contact (25), have led to consideration of an oral source of transmission. In a cohort of MSM from San Francisco, similar prevalences of KSHV infection from 1978 to 1996 were found, despite a reduction in HIV-1 seroprevalence and the institution of "safer" sexual practices (65). This raises the issue that sexual activity may be a marker for other types of intimate contact in this population. In keeping with this idea, PCR-based studies of the male genitourinary tract have described only infrequent, low-level shedding of KSHV DNA in genital secretions (22). Infectious virions have not been demonstrated in semen. Similarly, only infrequent and low-level shedding of KSHV DNA has been observed from male urethral and anorectal secretions (66). KSHV DNA has only rarely been detected in vaginal secretions (8, 46, 93), and heterosexual sex has not been clearly associated with KSHV transmission (76).
Evidence for nonsexual transmission is supported by data derived from studies in Africa, Italy, Egypt, and Japan which document KSHV infection in groups with very low risk of STI, including children (1, 8, 42, 91, 92). In regions of Africa where KSHV is endemic, a cumulative increase in KSHV seroprevalence from birth through adolescence has been observed, with more than 40% of children over 14 years of age infected and many infants seroconverting during the first year of life (2, 30, 56). Evidence for transmission through breast milk has not been found (7), and congenital infection is thought to be rare (54). The fact that children and other groups without risk of STI are infected with KSHV implies salivary transmission, as is seen with other human herpesviruses (HHVs), such as cytomegalovirus, HHV-6, HHV-7, and Epstein-Barr virus (EBV).
KSHV DNA has been detected frequently in human saliva (6, 44), and when found, it occurs at a titer 2 to 3 log units higher than those at other anatomic sites (66). Infectious KSHV has been documented in saliva (87). For these reasons, the oral cavity is unique in its ability to support lytic replication of KSHV. Published studies report that 11% to 68% of HIV-positive MSM infected with KSHV in the absence of KS shed viral DNA in saliva on at least one occasion (9, 22, 66). One study demonstrated that 39% of KSHV-seropositive MSM shed viral DNA from the oral cavity on more than a third of the days sampled, regardless of HIV-1 status (66). Current data indicate that KSHV does not replicate in salivary glands (21, 22). Epithelial cells lining the oral cavity are a probable source, given that nucleated oral epithelial cells from nonkeratinized mucosal surfaces have been shown to support viral infection in vivo, by the detection of KSHV-specific latent and lytic mRNA transcripts (66).
Other viruses, such as EBV (31, 49, 74, 96) and human papillomavirus (HPV) (reviewed in reference 51), undergo lytic viral replication in differentiating epithelia. The organotypic raft culture model, originally developed to study keratinocyte differentiation (70), has been of great value in the study of viral replication in developing epithelium (57). Organotypic raft cultures accurately reproduce the process of epithelial differentiation in vitro, beginning with undifferentiated keratinocyte monolayers that become confluent and polarized, forming tight junctions prior to the initiation of keratinocyte differentiation. A proportion of the keratinocytes remain in the basal layer and maintain proliferative potential, while other cells leave the basal layer, lose mitotic capability, and differentiate into mature epithelial cells, forming the multilayered structure of the epithelium (39). Keratinocytes follow a programmed pattern of gene expression during differentiation, each step of which is characterized by the expression of keratin, and keratin-associated, proteins (59). This in vitro model has demonstrated that HPV productively infects cutaneous and mucosal epithelia by a process that is tightly linked to epithelial differentiation (40).
In order to test the hypothesis that activation of KSHV lytic gene expression occurs during epithelial differentiation, we employed an organotypic raft culture model that utilized keratinocytes isolated from normal tonsils. We found that the process of epithelial differentiation activated KSHV lytic-gene expression and resulted in the production of infectious virus at the epithelial surface.
MATERIALS AND METHODS
Subjects. This study was approved by the Human Subjects Review Committee of the University of Washington. Oral and written informed consent was obtained from all participants. Healthy adults planning elective tonsillectomy were eligible to donate leftover tonsil tissues not required for routine pathology. All tonsillectomy procedures were performed by members of the Department of Otolaryngology at the University of Washington. There were no modifications of routine surgical practices and procedures. Subjects with tonsillar mass or asymmetry were excluded in case they had a carcinoma or lymphoma, but otherwise, there were no specific exclusion criteria.
Tonsil-derived organotypic raft cultures. Tonsil specimens were immediately transported in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Invitrogen Corporation) with 10% fetal bovine serum (FBS) (Gibco, Invitrogen Corporation) plus antibiotics (penicillin-streptomycin) at 4°C and processed on arrival. Tissue specimens were variable in size, with surface areas ranging from approximately 1 to 4 cm2. Epithelial cell sheets were separated from each specimen by careful dissection after overnight incubation in dispase II (24.0 U/ml; Gibco, Invitrogen Corporation) at 4°C. Collected materials were treated with 0.05% trypsin-EDTA (Gibco, Invitrogen Corporation) for 30 min at 37°C to form single-cell suspensions and plated at a concentration of 5 x 105 cells/ml in EpiLife medium (Ca2+; 0.06 M) supplemented with the HKGS kit (Cascade Biologics, Inc. Portland, OR) onto 60-mm culture dishes coated with mouse collagen type IV (BD Biosciences, Bedford, MA). The keratinocytes were placed in a 37°C CO2 incubator and fed every other day with EpiLife medium. The keratinocytes were expanded to 70% confluence and used for the organotypic tissue culture.
Autologous fibroblasts were recovered from each tonsil specimen by placing leftover pieces in a T75 flask and incubating them in DMEM supplemented with 10% FBS, L-glutamine, amphotericin B, ciprofloxacin, penicillin, streptomycin, and fresh vitamin C (50 μg/ml; Sigma, St. Louis, MO) at 37°C in a CO2 incubator and were fed every other day. Adherent fibroblasts were expanded and used to seed the bottoms of 12-well plates at a density of 105 cells/well for use in the raft culture model. Expanded keratinocyte cultures were trypsinized, washed, and seeded onto type I collagen-coated cell culture inserts (12-well format) at a density of 105 cells/insert in EpiLife medium. The inserts were incubated submerged at 37°C in a humidified 5% CO2 incubator for a period of 8 to 10 days until a confluent monolayer formed. The inserts were then transferred to a fresh well seeded with autologous fibroblasts at a density of 3 x 105 cells/well and submerged in FAD medium (three parts DMEM, one part Ham's F-12 [Gibco, Invitrogen Corporation], 10% FBS, 8.9 ng/ml epidermal growth factor [Sigma, St. Louis, MO], 0.45 ng/ml insulin [Sigma, St. Louis, MO], 0.45 ng/ml hydrocortisone [Sigma, St. Louis, MO], and 0.3 g/ml adenine [Sigma, St. Louis, MO]) for 24 h, followed by removal of the FAD medium from the upper chamber, exposing the keratinocyte layer to air. Once the layer was exposed, FAD medium (0.6 ml/well) was added to the lower chamber daily to ensure the insert was fed only from the bottom. The inserts were incubated at 37°C in a humidified 5% CO2 incubator for 6 days prior to harvest. Half of each insert was snap frozen fresh in Optimal Cutting Temperature medium (Sakura, Tokyo, Japan) and stored at –80°C. The remaining half was fixed in 4% paraformaldehyde for 30 min at room temperature (RT), snap frozen in Optimal Cutting Temperature medium, and stored at –80°C. A cryostat was used to cut 10-μm sections of tissue on silane-coated slides for further studies.
Three organotypic raft cultures (each derived from a separate donor in this study) were tested by PCR for the presence of herpes simplex virus types 1 and 2, varicella-zoster virus, cytomegalovirus, EBV, HHV-6, and high-risk HPV types (including types 6, 11, 16, 18, 31, 33, 35, 39, and 45) and found to be negative. Reference testing was performed by the Molecular Diagnostic Laboratory and the HPV Laboratory at the University of Washington using previously published methodology (5, 9, 43, 97).
Antibody detection of keratin proteins and viral ORF K8.1 late envelope glycoprotein. Frozen sections of organotypic raft cultures were thawed and washed in phosphate-buffered saline (PBS), pH 7.4. Tissue sections for keratin antibody staining were used directly for immunofluorescence histochemistry, whereas those used for detection of ORF K8.1 were air dried and fixed for 30 min in 4% paraformaldehyde at RT. Tissue sections were blocked with Serum-Free Protein Block (DAKO Corporation, Carpinteria, CA). Keratin antibodies were used to characterize organotypic raft cultures, including CK1, clone 34?B4; CK10, clone LHP1; CK4, clone 6B10; CK13, clone KS-1A3; CK5, clone XM26; CK14, clone LL002; CK6, clone LHK6B; CK16, clone LL025; CK19, clone b170; and involucrin, clone SY5,(all from NovoCastra Laboratories, Newcastle, United Kingdom), at dilutions recommended by the manufacturer. ORF K8.1 protein was detected with monoclonal antibody (MAb) A4 diluted 1:20 in PBS (98). Slide sections were incubated with primary antibody for 1 h at RT, washed, and detected with isotype-specific Qdot 655-conjugated goat F(ab')2 anti-mouse immunoglobulin G (IgG) secondary antibody (Quantum Dot Corporation, Hayward, CA) diluted 1:200 for 30 min at RT. Slides were mounted with Vectashield fluorescence mounting medium with DAPI (4',6'-diamidino-2-phenylindole) (Vector Laboratories, Inc., Burlingame, CA). Staining controls for keratin staining included omission of the primary antibody and normal human tonsil epithelium. Staining controls for the ORF K8.1 MAb included omission of the primary antibody, uninfected raft culture sections, and productively infected Vero cells as positive controls.
Antibody detection of viral ORF 73 (LANA) in rKSHV.219-infected oral keratinocytes. The immunofluorescence detection of KSHV ORF 73 protein was carried out with rat MAb to HHV-8 ORF 73 (ABI, Columbia, MD) diluted 1:100 in 10% normal goat serum. Alexa Fluor 647 goat anti-rat IgG (Molecular Probes, Eugene, OR) was used for fluorescence detection. Oral keratinocytes infected with recombinant KSHV.219 (rKSHV.219) for 10 days and split twice over that time were fixed for 30 min in 4% paraformaldehyde at RT, permeabilized with 0.5% Triton X-100 at RT for 15 min, and reacted with rat MAb to ORF 73. The keratinocytes were washed twice with PBS and reacted with Alexa Fluor 647 goat anti-rat IgG diluted 1:200 for 30 min, washed twice with PBS, counterstained with 1 μg/ml DAPI for 1 min, and washed in PBS.
rKSHV.219. rKSHV.219 is a JSC-derived recombinant KSHV that expresses the green fluorescent protein (GFP) during latent and lytic replication from the cellular promoter EF-1 and the red fluorescent protein (RFP) during lytic replication from the viral lytic PAN promoter. Details concerning the construction and production of rKSHV.219 have been previously published (89).
Infection of organotypic raft cultures. Epithelial cell monolayers were infected with rKSHV.219 on cell culture inserts at 70% confluence in EpiLife medium. Inserts were infected from the top with 200 μl of rKSHV.219 with Polybrene (7.5 μg/ml) and incubated at 37°C in a humidified 5% CO2 incubator for 2 h. A single preparation of virus was used for these experiments at a titer of 6 x 105 infectious units (IU)/ml as determined by titering on 293 cells and counting GFP-positive cells. Virus stock was removed, and the insert was washed with sterile PBS twice. The insert was again submerged in EpiLife medium until confluent and processed as described above. The inserts were observed daily for GFP and RFP expression by fluorescence microscopy as evidence of latent or lytic infection of cells.
Determination of IU of rKSHV.219. For the detection of infectious KSHV in the raft cultures, 0.3 ml of medium was placed in the upper chamber 5 days after air exposure and collected after overnight incubation. The determination of IU of rKSHV.219 was performed as previously described (89). Briefly, cells were removed from each supernatant by centrifugation (300 x g; 10 min) and then passed through a 0.45-μm filter and used as rKSHV.219 inocula for the infection of 293 cells. The number of IU of rKSHV.219 was then determined from the number of GFP-positive 293 cells counted 2 days postinfection. To maximize the infection, it was carried out in the presence of Polybrene (7.5 μg/ml) with centrifugation enhancement (450 x g; 20 min), and then the cells were incubated for 3 h before the medium was replaced with fresh medium without Polybrene. Medium supernatant from the rKSHV.219-infected epithelial cells was collected prior to air exposure and used as a control.
Microscopy. A Nikon Eclipse TE2000S inverted fluorescence microscope equipped with filter sets TE2000 DAPI, TE2000 FITC HYQ, TE2000 Texas Red HYQ, and Omega Optical XF305-2 was used. Images were acquired with a Photometrics CoolSNAP cf digital camera and Metavue Imaging software version 6.1.
RESULTS
Development of nonkeratinized stratified squamous epithelium in vitro. An in vitro organotypic raft culture model of oral epithelium was produced using oral keratinocytes isolated from human tonsils. The keratinocytes were expanded ex vivo and plated on cell culture inserts that served as the support for the organotypic culture. Autologous fibroblasts were incorporated into the model by seeding the bottom of each cell culture well. Confluent keratinocyte monolayers were exposed to air and observed daily prior to harvest on the sixth day. Morphological characteristics of the epithelium were assessed by phase microscopy, DAPI staining, and antibody detection of specific markers of epithelial differentiation. Raft cultures developed a multilayered epithelium 8 to 12 layers thick (Fig. 1). Nucleated cells were observed at the surface of the raft culture by DAPI staining (Fig. 1), a feature consistent with nonkeratinized epithelium (81, 85). Types of epithelia and the state of differentiation can be profiled by the expression pattern of keratins, a family of over 30 proteins expressed in specific pairings of type I (acidic) and type II (basic) members (59). To further characterize the in vitro-generated epithelium, cross sections were analyzed with monoclonal antibodies to specific keratins and involucrin. The keratin pair K4 and K13, specific for nonkeratinized oral stratified epithelia (19, 20, 59, 94), was detected in suprabasal layers (K13 is shown in Fig. 1). Conversely, the keratin pair K1 and K10, specifically found in keratinized epithelia (20, 47, 59, 75, 78), was absent (K1 is shown in Fig. 1). The keratin pair K5 and K14, which is found in all types of oral stratified epithelia (61, 67), was present in basal and suprabasal layers of the raft culture, consistent with the tissue of origin (K14 is shown in Fig. 1). Other keratin markers found in differentiated tonsil epithelium, including keratins K6 and K19 (35, 50, 58, 62), were also demonstrated in suprabasal layers of the raft culture (Fig. 1). Involucrin, a keratin-associated protein indicative of epithelial differentiation (41, 68, 83), was observed in the suprabasal layers of the raft culture (Fig. 1). These experiments demonstrated that the epithelial morphology and keratin expression pattern observed in our in vitro organotypic raft culture model was consistent with that of normal human tonsil epithelium (58).
KSHV replication in keratinocytes and in vitro epithelium. In vivo epithelium arises from keratinocytes (progenitor cells) located in the basal layer (39). Similarly, in vitro organotypic raft cultures are formed from undifferentiated keratinocyte monolayers. Therefore, oral keratinocytes, which were cultured to maintain an undifferentiated state, were infected with rKSHV.219 to examine latent and lytic replication. Figure 2A depicts undifferentiated, primary oral keratinocytes 10 days after infection with rKSHV.219 by phase (Fig. 2A, 1) and fluorescence for GFP (Fig. 2A, 2) and RFP (Fig. 2A, 3). No evidence of RFP expression was seen, indicative of latent viral infection (Fig. 2A, 3). In Fig. 2B, the same keratinocytes were examined for LANA expression by phase (Fig. 2B, 1) and by fluorescence for LANA, GFP, and DAPI (Fig. 2B, 2). GFP-expressing cells demonstrated punctate nuclear staining for LANA, while non-GFP-expressing cells did not. The absence of RFP expression and the presence of punctate nuclear LANA staining are indicative of latent viral infection in these undifferentiated keratinocyte monolayers.
Next, this model of oral epithelium was used to assess the impact of epithelial differentiation on KSHV replication. This was accomplished by infecting keratinocytes at 50 to 70% confluence on culture inserts with rKSHV.219 and using the infected keratinocytes to initiate organotypic raft cultures. The infected keratinocytes continued to grow, became confluent in 2 to 3 days, and expressed GFP but not RFP, as seen in an intact keratinocyte monolayer 1 day prior to air exposure (Fig. 3A). This was indicative of latent infection, as has been previously reported for KSHV-infected keratinocyte monolayers (10, 88). This was also similar to long-term cultures of rKSHV.219-infected keratinocytes that were passaged submerged in epithelial medium (Fig. 2). There is a single published report that describes lytic infection of primary epithelial cell monolayers in culture, but it is not currently known whether this observation was due to differences in the virus used or cell culture conditions (26). One day postconfluence, the medium from the top chamber was removed in order to expose the apical surfaces of the keratinocytes to air and to promote differentiation of the epithelium. Intact living cultures were examined daily for GFP and RFP expression. Images of phase, GFP expression, and RFP expression at 3 and 6 days after air exposure are shown (Fig. 3B and C). The expression of RFP from the lytic PAN promoter indicated that activation of lytic-gene expression occurred in the differentiating epithelium.
RFP expression in rKSHV.219-infected cells indicates early gene expression, because it is under the control of the lytic PAN promoter, which is directly activated by Rta (79). To determine if late lytic-gene expression occurred in these epithelial cells, cultures were harvested 6 days after air exposure and intact rafts were analyzed with antibody to the late viral glycoprotein K8.1. K8.1 expression was identified in a subset of cells expressing RFP (Fig. 4). In these cultures, not all cells with KSHV early gene expression progressed to late gene expression, an observation made in other cell types (18, 60, 89), although the reasons are not understood. These experiments demonstrated that the process of epithelial differentiation activated KSHV lytic gene expression. Observations of the intact organotypic raft culture suggested that this was predominately occurring in cells at the apical surface; therefore, the location of lytic-gene expression within the rafts was examined.
To identify the location of cells with lytic-gene expression within the epithelial tissue, rafts were sectioned and examined by fluorescence microscopy for GFP and RFP expression. On cross section, GFP was distributed throughout all layers of the raft culture epithelium but was expressed most intensely at the surface, where squames had become flattened and more densely compacted (Fig. 5B). RFP was observed primarily in cells at the apical surface of the raft culture, indicating that lytic-gene expression was occurring predominantly in differentiated epithelial cells (Fig. 5C). In order to compare the localization of late lytic-protein expression with that of early lytic-gene expression (indicated by RFP), sections of the raft cultures were stained with antibody to the ORF K8.1 protein. As seen for the RFP-positive cells, K8.1 was detected in cells at the apical surface of the epithelium (Fig. 6).
Production of infectious KSHV. Because the eventual outcome of the activation of KSHV lytic-gene expression is the generation of infectious virus, the organotypic raft cultures were tested for the production of infectious KSHV. This was carried out by adding medium to the upper chamber of the cultures 5 days after air exposure. The medium was removed after overnight incubation and used to inoculate 293 cells to determine the presence of IU of rKSHV.219 by examination for GFP-positive 293 cells 2 days postinoculation. Figure 7 shows an image of 293 cells infected with supernatant from one of the rKSHV.219-infected raft cultures. Three raft cultures derived from one tonsil and two from a second tonsil were tested, and all were found to produce infectious virus, ranging from 44 to 165 IU per 0.9-cm2 insert. Medium removed from the upper chamber at the time of air exposure was also used to inoculate 293 cells and was found to be negative. This demonstrated that differentiating keratinocytes activated KSHV lytic replication and became permissive for the production of infectious KSHV.
DISCUSSION
Saliva has been the only readily transmitted bodily fluid proven to contain infectious KSHV (87), and epidemiological data suggest saliva as a likely route of transmission (2, 30, 56), but the source of KSHV in saliva has not been well characterized. Unlike human cytomegalovirus or human herpesvirus 6, current data suggest that KSHV does not replicate in salivary glands (22, 66). EBV and HPV activate lytic replication in developing epithelia, suggesting that KSHV might also replicate in epithelial cells lining the oral cavity. In order to study KSHV replication in oral epithelia, we developed an organotypic raft culture model, derived from human tonsil keratinocytes, that recapitulated the characteristics of a nonkeratinized stratified squamous oral epithelium. Using this model, we examined the impact of epithelial differentiation on KSHV replication. We found that undifferentiated primary keratinocytes maintained latent infection with KSHV; however, the process of epithelial differentiation resulted in the activation of both early and late lytic viral gene expression, with the release of infectious KSHV at the apical surface of the epithelium.
The production of a nonkeratinized organotypic raft culture model of oral epithelium was a critical component of these studies. The organotypic raft culture model is a well-established system for the production of epithelia in vitro. Early raft culture models, which utilized skin keratinocytes to produce multilayered epithelial tissue, exhibited differentiation patterns that resembled the tissue from which the keratinocytes originated (3, 4, 95). Keratinocytes were cultured on dermal equivalents composed of type I collagen and fibroblasts and raised to the air-medium interface to promote differentiation. This model has also been applied to keratinocytes derived from the oral cavity, including buccal (16, 17, 33, 34, 73), lingual (38), and peritonsillar (62) surfaces. Several of these studies underscored the importance of fibroblasts located in adjacent connective tissue, which provide permissive and instructive signals to differentiating keratinocytes (64, 77). In our system, primary keratinocytes isolated from nonkeratinized tonsil epithelium were seeded onto semipermeable cell culture inserts, and a fibroblast feeder layer derived from the tonsil dermis was established in the cell culture well beneath the insert. This format allowed excellent visualization and photography of cultures during development and provided a stable support for histological sectioning of harvested tissue. Raft cultures derived from primary tonsil keratinocytes had the expected morphological characteristics of nonkeratinized oral epithelium (58). An advanced stage of epithelial differentiation was achieved in suprabasal layers of the raft culture, as evidenced by expression of keratins 4 and 13 (59, 94) and involucrin (68). In addition, the presence of nucleated cells at the apical surface, the absence of specific markers of keratinization (keratins 1 and 10), and suprabasal expression of specific markers for nonkeratinized stratified squamous epithelium (keratins 4 and 13) (47) showed this to be an appropriate system to examine KSHV replication in oral epithelia.
We used our in vitro raft culture model of oral epithelium to test the hypothesis that basal keratinocytes, latently infected with KSHV, would activate viral lytic cycle gene expression during the process of epithelial differentiation, as suggested for the activation of EBV in the oral epithelia (49, 96). EBV, a gammaherpesvirus related to KSHV, is found in B cells and epithelial cells and is transmitted in saliva. Although the role of epithelial cells in primary EBV infection remains controversial (37, 63, 74), suprabasal epithelial cells in oral hairy leukoplakia lesions have been shown to replicate viral genomes and to express both lytic (36, 96) and latent (86) cycle antigens. Of value in our study, KSHV readily infected primary oral keratinocytes in culture and established a latent infection, which allowed us to initiate the process of epithelial differentiation using rKSHV.219-infected primary keratinocytes. The use of rKSHV.219 allowed the daily observation of raft cultures during differentiation for the identification of infected cells by GFP expression and for the activation of lytic gene expression indicated by the expression of RFP. While no RFP was seen in the basal layer of the epithelium, the process of keratinocyte differentiation resulted in the expression of RFP by many cells at the upper layer of the culture. While the expression of RFP from the lytic PAN promoter indicates early gene expression, a subset of RFP-positive cells was found to have late lytic gene expression as well, indicated by the presence of the late lytic envelope glycoprotein K8.1. The expression of K8.1 requires viral lytic DNA synthesis and therefore signifies that keratinocyte differentiation activates lytic DNA replication (13). The result of lytic replication is the production of infectious virus. In this in vitro model of oral epithelium, infectious virus was recovered from the apical surface of the raft culture, demonstrating that the complete KSHV lytic replication cycle can be completed as latently infected keratinocytes differentiate into a mature epithelium. Unlike in vitro epithelial models of EBV (49) and HPV (57) infection, which require stimulation with a phorbol ester to induce viral lytic replication, the activation of KSHV lytic-gene expression in differentiating primary keratinocytes is brought about by a natural cellular process.
KSHV lytic-cycle activation is initiated by Rta, the immediate-early transcriptional activator, expressed from open reading frame (ORF) 50 (53, 84, 90). Other agents known to induce KSHV lytic-cycle activation include phorbol esters (12, 69) and DNA-demethylating agents, such as azacytidine (15). Unlike KSHV culture systems that rely on the chemical treatment of cells, activation of KSHV lytic-gene expression in raft culture epithelium is brought about by a natural cellular process. Although the mechanism is unknown, one possible explanation is chromatin remodeling. Chromatin rearrangement around the late promoter region during epithelial differentiation has been described for HPV (23) and suggests that DNA binding of differentiation-specific cellular transcription factors is involved. Transcriptional regulation in eukaryotic cells is heavily dependent upon histone acetylation and methylation (reviewed in reference 24). Keratinocyte differentiation and growth arrest can be induced by inhibitors of histone deacetylase, such as sodium butyrate (72, 82) and tricostatin A (71). The KSHV Rta promoter can also be induced by these histone deacetylase inhibitors (52, 89), and Rta itself is involved in the recruitment of cellular factors (32, 35) that influence chromatin remodeling and access to cellular transcription factors (52). Another possible mechanism is the recruitment of cellular transcription factors at gene promoter sites by regulation of AP1, Sp1, and CCATT/enhancer-binding proteins (27, 28). ORF 50 utilizes a GC box initiator element and binds Sp family transcription factors (52), as do other cellular and viral promoters (48). Sp1 proteins are critical transactivators of regulated keratinocyte gene expression (27), and promoters containing GC box initiator elements may be more active in keratinocytes. For example, the EBV early protein BMRF2, which utilizes a GC box initiator element and is highly expressed in oral hairy leukoplakia lesions, exhibits 10-fold-greater activity in epithelial cells than in lymphocytes (45). The mechanisms of activation (chromatin remodeling and recruitment of cellular transcription factors) are not mutually exclusive, and other factors could be involved. Further experiments will be required to determine the mechanisms that are important for viral activation in differentiating cells.
In summary, this work demonstrates that the process of oral epithelial differentiation can activate KSHV lytic-gene expression. Moreover, because differentiated oral keratinocytes are competent to support productive viral replication with the release of infectious viral particles, oral epithelial cells are a likely source of infectious KSHV present in saliva. The switch between latent and lytic replication is the hallmark of herpesvirus biology, allowing the permanent infection of the host, and is critical to the pathogenesis and transmission of herpesviruses. The results presented here illustrate an in vitro model of a natural cellular process causing the switch from latent to lytic replication and will be of value in unraveling the cellular and viral mechanisms involved in activation of KSHV from latency.
ACKNOWLEDGMENTS
We thank Bala Chandran for antibodies to KSHV K8.1 viral protein; Meei Li Huang, Nancy Kiviat, and Donna Kenney for viral PCR testing; and Negin Nowbarn-Nekahi for critical reading of the manuscript.
This work was supported by Public Health Service Grants DE14149-04 to J.V. and AI51946-02 to A.S.J.
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Otolaryngology, University of Washington, Seattle, Washington 98195
ABSTRACT
The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1 promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.
INTRODUCTION
Since its initial description over a decade ago (14), Kaposi's sarcoma-associated herpesvirus (KSHV) has been recognized as a significant viral pathogen, particularly for immunocompromised hosts and persons infected with human immunodeficiency virus type 1 (HIV-1). KSHV is the etiologic agent of Kaposi's sarcoma (KS), having been demonstrated in biopsy specimens of all forms of KS despite distinct differences in the geographic origin, age, and gender of affected persons (29). KSHV is also associated with multicentric Castleman's disease (80) and primary effusion lymphoma (11).
The precise modes of KSHV transmission are not clearly defined; however, epidemiological data suggest that both sexual and nonsexual routes are possible. The association between KSHV transmission and sexual activity has been largely defined in North American men who have sex with men (MSM), where risk factors include sex with a partner who has KS (66), a history of sexually transmitted infections (STIs) (55), and an increased number of sexual partners (55). Other risk factors, however, such as deep kissing with an HIV-positive partner (66) and orogenital contact (25), have led to consideration of an oral source of transmission. In a cohort of MSM from San Francisco, similar prevalences of KSHV infection from 1978 to 1996 were found, despite a reduction in HIV-1 seroprevalence and the institution of "safer" sexual practices (65). This raises the issue that sexual activity may be a marker for other types of intimate contact in this population. In keeping with this idea, PCR-based studies of the male genitourinary tract have described only infrequent, low-level shedding of KSHV DNA in genital secretions (22). Infectious virions have not been demonstrated in semen. Similarly, only infrequent and low-level shedding of KSHV DNA has been observed from male urethral and anorectal secretions (66). KSHV DNA has only rarely been detected in vaginal secretions (8, 46, 93), and heterosexual sex has not been clearly associated with KSHV transmission (76).
Evidence for nonsexual transmission is supported by data derived from studies in Africa, Italy, Egypt, and Japan which document KSHV infection in groups with very low risk of STI, including children (1, 8, 42, 91, 92). In regions of Africa where KSHV is endemic, a cumulative increase in KSHV seroprevalence from birth through adolescence has been observed, with more than 40% of children over 14 years of age infected and many infants seroconverting during the first year of life (2, 30, 56). Evidence for transmission through breast milk has not been found (7), and congenital infection is thought to be rare (54). The fact that children and other groups without risk of STI are infected with KSHV implies salivary transmission, as is seen with other human herpesviruses (HHVs), such as cytomegalovirus, HHV-6, HHV-7, and Epstein-Barr virus (EBV).
KSHV DNA has been detected frequently in human saliva (6, 44), and when found, it occurs at a titer 2 to 3 log units higher than those at other anatomic sites (66). Infectious KSHV has been documented in saliva (87). For these reasons, the oral cavity is unique in its ability to support lytic replication of KSHV. Published studies report that 11% to 68% of HIV-positive MSM infected with KSHV in the absence of KS shed viral DNA in saliva on at least one occasion (9, 22, 66). One study demonstrated that 39% of KSHV-seropositive MSM shed viral DNA from the oral cavity on more than a third of the days sampled, regardless of HIV-1 status (66). Current data indicate that KSHV does not replicate in salivary glands (21, 22). Epithelial cells lining the oral cavity are a probable source, given that nucleated oral epithelial cells from nonkeratinized mucosal surfaces have been shown to support viral infection in vivo, by the detection of KSHV-specific latent and lytic mRNA transcripts (66).
Other viruses, such as EBV (31, 49, 74, 96) and human papillomavirus (HPV) (reviewed in reference 51), undergo lytic viral replication in differentiating epithelia. The organotypic raft culture model, originally developed to study keratinocyte differentiation (70), has been of great value in the study of viral replication in developing epithelium (57). Organotypic raft cultures accurately reproduce the process of epithelial differentiation in vitro, beginning with undifferentiated keratinocyte monolayers that become confluent and polarized, forming tight junctions prior to the initiation of keratinocyte differentiation. A proportion of the keratinocytes remain in the basal layer and maintain proliferative potential, while other cells leave the basal layer, lose mitotic capability, and differentiate into mature epithelial cells, forming the multilayered structure of the epithelium (39). Keratinocytes follow a programmed pattern of gene expression during differentiation, each step of which is characterized by the expression of keratin, and keratin-associated, proteins (59). This in vitro model has demonstrated that HPV productively infects cutaneous and mucosal epithelia by a process that is tightly linked to epithelial differentiation (40).
In order to test the hypothesis that activation of KSHV lytic gene expression occurs during epithelial differentiation, we employed an organotypic raft culture model that utilized keratinocytes isolated from normal tonsils. We found that the process of epithelial differentiation activated KSHV lytic-gene expression and resulted in the production of infectious virus at the epithelial surface.
MATERIALS AND METHODS
Subjects. This study was approved by the Human Subjects Review Committee of the University of Washington. Oral and written informed consent was obtained from all participants. Healthy adults planning elective tonsillectomy were eligible to donate leftover tonsil tissues not required for routine pathology. All tonsillectomy procedures were performed by members of the Department of Otolaryngology at the University of Washington. There were no modifications of routine surgical practices and procedures. Subjects with tonsillar mass or asymmetry were excluded in case they had a carcinoma or lymphoma, but otherwise, there were no specific exclusion criteria.
Tonsil-derived organotypic raft cultures. Tonsil specimens were immediately transported in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Invitrogen Corporation) with 10% fetal bovine serum (FBS) (Gibco, Invitrogen Corporation) plus antibiotics (penicillin-streptomycin) at 4°C and processed on arrival. Tissue specimens were variable in size, with surface areas ranging from approximately 1 to 4 cm2. Epithelial cell sheets were separated from each specimen by careful dissection after overnight incubation in dispase II (24.0 U/ml; Gibco, Invitrogen Corporation) at 4°C. Collected materials were treated with 0.05% trypsin-EDTA (Gibco, Invitrogen Corporation) for 30 min at 37°C to form single-cell suspensions and plated at a concentration of 5 x 105 cells/ml in EpiLife medium (Ca2+; 0.06 M) supplemented with the HKGS kit (Cascade Biologics, Inc. Portland, OR) onto 60-mm culture dishes coated with mouse collagen type IV (BD Biosciences, Bedford, MA). The keratinocytes were placed in a 37°C CO2 incubator and fed every other day with EpiLife medium. The keratinocytes were expanded to 70% confluence and used for the organotypic tissue culture.
Autologous fibroblasts were recovered from each tonsil specimen by placing leftover pieces in a T75 flask and incubating them in DMEM supplemented with 10% FBS, L-glutamine, amphotericin B, ciprofloxacin, penicillin, streptomycin, and fresh vitamin C (50 μg/ml; Sigma, St. Louis, MO) at 37°C in a CO2 incubator and were fed every other day. Adherent fibroblasts were expanded and used to seed the bottoms of 12-well plates at a density of 105 cells/well for use in the raft culture model. Expanded keratinocyte cultures were trypsinized, washed, and seeded onto type I collagen-coated cell culture inserts (12-well format) at a density of 105 cells/insert in EpiLife medium. The inserts were incubated submerged at 37°C in a humidified 5% CO2 incubator for a period of 8 to 10 days until a confluent monolayer formed. The inserts were then transferred to a fresh well seeded with autologous fibroblasts at a density of 3 x 105 cells/well and submerged in FAD medium (three parts DMEM, one part Ham's F-12 [Gibco, Invitrogen Corporation], 10% FBS, 8.9 ng/ml epidermal growth factor [Sigma, St. Louis, MO], 0.45 ng/ml insulin [Sigma, St. Louis, MO], 0.45 ng/ml hydrocortisone [Sigma, St. Louis, MO], and 0.3 g/ml adenine [Sigma, St. Louis, MO]) for 24 h, followed by removal of the FAD medium from the upper chamber, exposing the keratinocyte layer to air. Once the layer was exposed, FAD medium (0.6 ml/well) was added to the lower chamber daily to ensure the insert was fed only from the bottom. The inserts were incubated at 37°C in a humidified 5% CO2 incubator for 6 days prior to harvest. Half of each insert was snap frozen fresh in Optimal Cutting Temperature medium (Sakura, Tokyo, Japan) and stored at –80°C. The remaining half was fixed in 4% paraformaldehyde for 30 min at room temperature (RT), snap frozen in Optimal Cutting Temperature medium, and stored at –80°C. A cryostat was used to cut 10-μm sections of tissue on silane-coated slides for further studies.
Three organotypic raft cultures (each derived from a separate donor in this study) were tested by PCR for the presence of herpes simplex virus types 1 and 2, varicella-zoster virus, cytomegalovirus, EBV, HHV-6, and high-risk HPV types (including types 6, 11, 16, 18, 31, 33, 35, 39, and 45) and found to be negative. Reference testing was performed by the Molecular Diagnostic Laboratory and the HPV Laboratory at the University of Washington using previously published methodology (5, 9, 43, 97).
Antibody detection of keratin proteins and viral ORF K8.1 late envelope glycoprotein. Frozen sections of organotypic raft cultures were thawed and washed in phosphate-buffered saline (PBS), pH 7.4. Tissue sections for keratin antibody staining were used directly for immunofluorescence histochemistry, whereas those used for detection of ORF K8.1 were air dried and fixed for 30 min in 4% paraformaldehyde at RT. Tissue sections were blocked with Serum-Free Protein Block (DAKO Corporation, Carpinteria, CA). Keratin antibodies were used to characterize organotypic raft cultures, including CK1, clone 34?B4; CK10, clone LHP1; CK4, clone 6B10; CK13, clone KS-1A3; CK5, clone XM26; CK14, clone LL002; CK6, clone LHK6B; CK16, clone LL025; CK19, clone b170; and involucrin, clone SY5,(all from NovoCastra Laboratories, Newcastle, United Kingdom), at dilutions recommended by the manufacturer. ORF K8.1 protein was detected with monoclonal antibody (MAb) A4 diluted 1:20 in PBS (98). Slide sections were incubated with primary antibody for 1 h at RT, washed, and detected with isotype-specific Qdot 655-conjugated goat F(ab')2 anti-mouse immunoglobulin G (IgG) secondary antibody (Quantum Dot Corporation, Hayward, CA) diluted 1:200 for 30 min at RT. Slides were mounted with Vectashield fluorescence mounting medium with DAPI (4',6'-diamidino-2-phenylindole) (Vector Laboratories, Inc., Burlingame, CA). Staining controls for keratin staining included omission of the primary antibody and normal human tonsil epithelium. Staining controls for the ORF K8.1 MAb included omission of the primary antibody, uninfected raft culture sections, and productively infected Vero cells as positive controls.
Antibody detection of viral ORF 73 (LANA) in rKSHV.219-infected oral keratinocytes. The immunofluorescence detection of KSHV ORF 73 protein was carried out with rat MAb to HHV-8 ORF 73 (ABI, Columbia, MD) diluted 1:100 in 10% normal goat serum. Alexa Fluor 647 goat anti-rat IgG (Molecular Probes, Eugene, OR) was used for fluorescence detection. Oral keratinocytes infected with recombinant KSHV.219 (rKSHV.219) for 10 days and split twice over that time were fixed for 30 min in 4% paraformaldehyde at RT, permeabilized with 0.5% Triton X-100 at RT for 15 min, and reacted with rat MAb to ORF 73. The keratinocytes were washed twice with PBS and reacted with Alexa Fluor 647 goat anti-rat IgG diluted 1:200 for 30 min, washed twice with PBS, counterstained with 1 μg/ml DAPI for 1 min, and washed in PBS.
rKSHV.219. rKSHV.219 is a JSC-derived recombinant KSHV that expresses the green fluorescent protein (GFP) during latent and lytic replication from the cellular promoter EF-1 and the red fluorescent protein (RFP) during lytic replication from the viral lytic PAN promoter. Details concerning the construction and production of rKSHV.219 have been previously published (89).
Infection of organotypic raft cultures. Epithelial cell monolayers were infected with rKSHV.219 on cell culture inserts at 70% confluence in EpiLife medium. Inserts were infected from the top with 200 μl of rKSHV.219 with Polybrene (7.5 μg/ml) and incubated at 37°C in a humidified 5% CO2 incubator for 2 h. A single preparation of virus was used for these experiments at a titer of 6 x 105 infectious units (IU)/ml as determined by titering on 293 cells and counting GFP-positive cells. Virus stock was removed, and the insert was washed with sterile PBS twice. The insert was again submerged in EpiLife medium until confluent and processed as described above. The inserts were observed daily for GFP and RFP expression by fluorescence microscopy as evidence of latent or lytic infection of cells.
Determination of IU of rKSHV.219. For the detection of infectious KSHV in the raft cultures, 0.3 ml of medium was placed in the upper chamber 5 days after air exposure and collected after overnight incubation. The determination of IU of rKSHV.219 was performed as previously described (89). Briefly, cells were removed from each supernatant by centrifugation (300 x g; 10 min) and then passed through a 0.45-μm filter and used as rKSHV.219 inocula for the infection of 293 cells. The number of IU of rKSHV.219 was then determined from the number of GFP-positive 293 cells counted 2 days postinfection. To maximize the infection, it was carried out in the presence of Polybrene (7.5 μg/ml) with centrifugation enhancement (450 x g; 20 min), and then the cells were incubated for 3 h before the medium was replaced with fresh medium without Polybrene. Medium supernatant from the rKSHV.219-infected epithelial cells was collected prior to air exposure and used as a control.
Microscopy. A Nikon Eclipse TE2000S inverted fluorescence microscope equipped with filter sets TE2000 DAPI, TE2000 FITC HYQ, TE2000 Texas Red HYQ, and Omega Optical XF305-2 was used. Images were acquired with a Photometrics CoolSNAP cf digital camera and Metavue Imaging software version 6.1.
RESULTS
Development of nonkeratinized stratified squamous epithelium in vitro. An in vitro organotypic raft culture model of oral epithelium was produced using oral keratinocytes isolated from human tonsils. The keratinocytes were expanded ex vivo and plated on cell culture inserts that served as the support for the organotypic culture. Autologous fibroblasts were incorporated into the model by seeding the bottom of each cell culture well. Confluent keratinocyte monolayers were exposed to air and observed daily prior to harvest on the sixth day. Morphological characteristics of the epithelium were assessed by phase microscopy, DAPI staining, and antibody detection of specific markers of epithelial differentiation. Raft cultures developed a multilayered epithelium 8 to 12 layers thick (Fig. 1). Nucleated cells were observed at the surface of the raft culture by DAPI staining (Fig. 1), a feature consistent with nonkeratinized epithelium (81, 85). Types of epithelia and the state of differentiation can be profiled by the expression pattern of keratins, a family of over 30 proteins expressed in specific pairings of type I (acidic) and type II (basic) members (59). To further characterize the in vitro-generated epithelium, cross sections were analyzed with monoclonal antibodies to specific keratins and involucrin. The keratin pair K4 and K13, specific for nonkeratinized oral stratified epithelia (19, 20, 59, 94), was detected in suprabasal layers (K13 is shown in Fig. 1). Conversely, the keratin pair K1 and K10, specifically found in keratinized epithelia (20, 47, 59, 75, 78), was absent (K1 is shown in Fig. 1). The keratin pair K5 and K14, which is found in all types of oral stratified epithelia (61, 67), was present in basal and suprabasal layers of the raft culture, consistent with the tissue of origin (K14 is shown in Fig. 1). Other keratin markers found in differentiated tonsil epithelium, including keratins K6 and K19 (35, 50, 58, 62), were also demonstrated in suprabasal layers of the raft culture (Fig. 1). Involucrin, a keratin-associated protein indicative of epithelial differentiation (41, 68, 83), was observed in the suprabasal layers of the raft culture (Fig. 1). These experiments demonstrated that the epithelial morphology and keratin expression pattern observed in our in vitro organotypic raft culture model was consistent with that of normal human tonsil epithelium (58).
KSHV replication in keratinocytes and in vitro epithelium. In vivo epithelium arises from keratinocytes (progenitor cells) located in the basal layer (39). Similarly, in vitro organotypic raft cultures are formed from undifferentiated keratinocyte monolayers. Therefore, oral keratinocytes, which were cultured to maintain an undifferentiated state, were infected with rKSHV.219 to examine latent and lytic replication. Figure 2A depicts undifferentiated, primary oral keratinocytes 10 days after infection with rKSHV.219 by phase (Fig. 2A, 1) and fluorescence for GFP (Fig. 2A, 2) and RFP (Fig. 2A, 3). No evidence of RFP expression was seen, indicative of latent viral infection (Fig. 2A, 3). In Fig. 2B, the same keratinocytes were examined for LANA expression by phase (Fig. 2B, 1) and by fluorescence for LANA, GFP, and DAPI (Fig. 2B, 2). GFP-expressing cells demonstrated punctate nuclear staining for LANA, while non-GFP-expressing cells did not. The absence of RFP expression and the presence of punctate nuclear LANA staining are indicative of latent viral infection in these undifferentiated keratinocyte monolayers.
Next, this model of oral epithelium was used to assess the impact of epithelial differentiation on KSHV replication. This was accomplished by infecting keratinocytes at 50 to 70% confluence on culture inserts with rKSHV.219 and using the infected keratinocytes to initiate organotypic raft cultures. The infected keratinocytes continued to grow, became confluent in 2 to 3 days, and expressed GFP but not RFP, as seen in an intact keratinocyte monolayer 1 day prior to air exposure (Fig. 3A). This was indicative of latent infection, as has been previously reported for KSHV-infected keratinocyte monolayers (10, 88). This was also similar to long-term cultures of rKSHV.219-infected keratinocytes that were passaged submerged in epithelial medium (Fig. 2). There is a single published report that describes lytic infection of primary epithelial cell monolayers in culture, but it is not currently known whether this observation was due to differences in the virus used or cell culture conditions (26). One day postconfluence, the medium from the top chamber was removed in order to expose the apical surfaces of the keratinocytes to air and to promote differentiation of the epithelium. Intact living cultures were examined daily for GFP and RFP expression. Images of phase, GFP expression, and RFP expression at 3 and 6 days after air exposure are shown (Fig. 3B and C). The expression of RFP from the lytic PAN promoter indicated that activation of lytic-gene expression occurred in the differentiating epithelium.
RFP expression in rKSHV.219-infected cells indicates early gene expression, because it is under the control of the lytic PAN promoter, which is directly activated by Rta (79). To determine if late lytic-gene expression occurred in these epithelial cells, cultures were harvested 6 days after air exposure and intact rafts were analyzed with antibody to the late viral glycoprotein K8.1. K8.1 expression was identified in a subset of cells expressing RFP (Fig. 4). In these cultures, not all cells with KSHV early gene expression progressed to late gene expression, an observation made in other cell types (18, 60, 89), although the reasons are not understood. These experiments demonstrated that the process of epithelial differentiation activated KSHV lytic gene expression. Observations of the intact organotypic raft culture suggested that this was predominately occurring in cells at the apical surface; therefore, the location of lytic-gene expression within the rafts was examined.
To identify the location of cells with lytic-gene expression within the epithelial tissue, rafts were sectioned and examined by fluorescence microscopy for GFP and RFP expression. On cross section, GFP was distributed throughout all layers of the raft culture epithelium but was expressed most intensely at the surface, where squames had become flattened and more densely compacted (Fig. 5B). RFP was observed primarily in cells at the apical surface of the raft culture, indicating that lytic-gene expression was occurring predominantly in differentiated epithelial cells (Fig. 5C). In order to compare the localization of late lytic-protein expression with that of early lytic-gene expression (indicated by RFP), sections of the raft cultures were stained with antibody to the ORF K8.1 protein. As seen for the RFP-positive cells, K8.1 was detected in cells at the apical surface of the epithelium (Fig. 6).
Production of infectious KSHV. Because the eventual outcome of the activation of KSHV lytic-gene expression is the generation of infectious virus, the organotypic raft cultures were tested for the production of infectious KSHV. This was carried out by adding medium to the upper chamber of the cultures 5 days after air exposure. The medium was removed after overnight incubation and used to inoculate 293 cells to determine the presence of IU of rKSHV.219 by examination for GFP-positive 293 cells 2 days postinoculation. Figure 7 shows an image of 293 cells infected with supernatant from one of the rKSHV.219-infected raft cultures. Three raft cultures derived from one tonsil and two from a second tonsil were tested, and all were found to produce infectious virus, ranging from 44 to 165 IU per 0.9-cm2 insert. Medium removed from the upper chamber at the time of air exposure was also used to inoculate 293 cells and was found to be negative. This demonstrated that differentiating keratinocytes activated KSHV lytic replication and became permissive for the production of infectious KSHV.
DISCUSSION
Saliva has been the only readily transmitted bodily fluid proven to contain infectious KSHV (87), and epidemiological data suggest saliva as a likely route of transmission (2, 30, 56), but the source of KSHV in saliva has not been well characterized. Unlike human cytomegalovirus or human herpesvirus 6, current data suggest that KSHV does not replicate in salivary glands (22, 66). EBV and HPV activate lytic replication in developing epithelia, suggesting that KSHV might also replicate in epithelial cells lining the oral cavity. In order to study KSHV replication in oral epithelia, we developed an organotypic raft culture model, derived from human tonsil keratinocytes, that recapitulated the characteristics of a nonkeratinized stratified squamous oral epithelium. Using this model, we examined the impact of epithelial differentiation on KSHV replication. We found that undifferentiated primary keratinocytes maintained latent infection with KSHV; however, the process of epithelial differentiation resulted in the activation of both early and late lytic viral gene expression, with the release of infectious KSHV at the apical surface of the epithelium.
The production of a nonkeratinized organotypic raft culture model of oral epithelium was a critical component of these studies. The organotypic raft culture model is a well-established system for the production of epithelia in vitro. Early raft culture models, which utilized skin keratinocytes to produce multilayered epithelial tissue, exhibited differentiation patterns that resembled the tissue from which the keratinocytes originated (3, 4, 95). Keratinocytes were cultured on dermal equivalents composed of type I collagen and fibroblasts and raised to the air-medium interface to promote differentiation. This model has also been applied to keratinocytes derived from the oral cavity, including buccal (16, 17, 33, 34, 73), lingual (38), and peritonsillar (62) surfaces. Several of these studies underscored the importance of fibroblasts located in adjacent connective tissue, which provide permissive and instructive signals to differentiating keratinocytes (64, 77). In our system, primary keratinocytes isolated from nonkeratinized tonsil epithelium were seeded onto semipermeable cell culture inserts, and a fibroblast feeder layer derived from the tonsil dermis was established in the cell culture well beneath the insert. This format allowed excellent visualization and photography of cultures during development and provided a stable support for histological sectioning of harvested tissue. Raft cultures derived from primary tonsil keratinocytes had the expected morphological characteristics of nonkeratinized oral epithelium (58). An advanced stage of epithelial differentiation was achieved in suprabasal layers of the raft culture, as evidenced by expression of keratins 4 and 13 (59, 94) and involucrin (68). In addition, the presence of nucleated cells at the apical surface, the absence of specific markers of keratinization (keratins 1 and 10), and suprabasal expression of specific markers for nonkeratinized stratified squamous epithelium (keratins 4 and 13) (47) showed this to be an appropriate system to examine KSHV replication in oral epithelia.
We used our in vitro raft culture model of oral epithelium to test the hypothesis that basal keratinocytes, latently infected with KSHV, would activate viral lytic cycle gene expression during the process of epithelial differentiation, as suggested for the activation of EBV in the oral epithelia (49, 96). EBV, a gammaherpesvirus related to KSHV, is found in B cells and epithelial cells and is transmitted in saliva. Although the role of epithelial cells in primary EBV infection remains controversial (37, 63, 74), suprabasal epithelial cells in oral hairy leukoplakia lesions have been shown to replicate viral genomes and to express both lytic (36, 96) and latent (86) cycle antigens. Of value in our study, KSHV readily infected primary oral keratinocytes in culture and established a latent infection, which allowed us to initiate the process of epithelial differentiation using rKSHV.219-infected primary keratinocytes. The use of rKSHV.219 allowed the daily observation of raft cultures during differentiation for the identification of infected cells by GFP expression and for the activation of lytic gene expression indicated by the expression of RFP. While no RFP was seen in the basal layer of the epithelium, the process of keratinocyte differentiation resulted in the expression of RFP by many cells at the upper layer of the culture. While the expression of RFP from the lytic PAN promoter indicates early gene expression, a subset of RFP-positive cells was found to have late lytic gene expression as well, indicated by the presence of the late lytic envelope glycoprotein K8.1. The expression of K8.1 requires viral lytic DNA synthesis and therefore signifies that keratinocyte differentiation activates lytic DNA replication (13). The result of lytic replication is the production of infectious virus. In this in vitro model of oral epithelium, infectious virus was recovered from the apical surface of the raft culture, demonstrating that the complete KSHV lytic replication cycle can be completed as latently infected keratinocytes differentiate into a mature epithelium. Unlike in vitro epithelial models of EBV (49) and HPV (57) infection, which require stimulation with a phorbol ester to induce viral lytic replication, the activation of KSHV lytic-gene expression in differentiating primary keratinocytes is brought about by a natural cellular process.
KSHV lytic-cycle activation is initiated by Rta, the immediate-early transcriptional activator, expressed from open reading frame (ORF) 50 (53, 84, 90). Other agents known to induce KSHV lytic-cycle activation include phorbol esters (12, 69) and DNA-demethylating agents, such as azacytidine (15). Unlike KSHV culture systems that rely on the chemical treatment of cells, activation of KSHV lytic-gene expression in raft culture epithelium is brought about by a natural cellular process. Although the mechanism is unknown, one possible explanation is chromatin remodeling. Chromatin rearrangement around the late promoter region during epithelial differentiation has been described for HPV (23) and suggests that DNA binding of differentiation-specific cellular transcription factors is involved. Transcriptional regulation in eukaryotic cells is heavily dependent upon histone acetylation and methylation (reviewed in reference 24). Keratinocyte differentiation and growth arrest can be induced by inhibitors of histone deacetylase, such as sodium butyrate (72, 82) and tricostatin A (71). The KSHV Rta promoter can also be induced by these histone deacetylase inhibitors (52, 89), and Rta itself is involved in the recruitment of cellular factors (32, 35) that influence chromatin remodeling and access to cellular transcription factors (52). Another possible mechanism is the recruitment of cellular transcription factors at gene promoter sites by regulation of AP1, Sp1, and CCATT/enhancer-binding proteins (27, 28). ORF 50 utilizes a GC box initiator element and binds Sp family transcription factors (52), as do other cellular and viral promoters (48). Sp1 proteins are critical transactivators of regulated keratinocyte gene expression (27), and promoters containing GC box initiator elements may be more active in keratinocytes. For example, the EBV early protein BMRF2, which utilizes a GC box initiator element and is highly expressed in oral hairy leukoplakia lesions, exhibits 10-fold-greater activity in epithelial cells than in lymphocytes (45). The mechanisms of activation (chromatin remodeling and recruitment of cellular transcription factors) are not mutually exclusive, and other factors could be involved. Further experiments will be required to determine the mechanisms that are important for viral activation in differentiating cells.
In summary, this work demonstrates that the process of oral epithelial differentiation can activate KSHV lytic-gene expression. Moreover, because differentiated oral keratinocytes are competent to support productive viral replication with the release of infectious viral particles, oral epithelial cells are a likely source of infectious KSHV present in saliva. The switch between latent and lytic replication is the hallmark of herpesvirus biology, allowing the permanent infection of the host, and is critical to the pathogenesis and transmission of herpesviruses. The results presented here illustrate an in vitro model of a natural cellular process causing the switch from latent to lytic replication and will be of value in unraveling the cellular and viral mechanisms involved in activation of KSHV from latency.
ACKNOWLEDGMENTS
We thank Bala Chandran for antibodies to KSHV K8.1 viral protein; Meei Li Huang, Nancy Kiviat, and Donna Kenney for viral PCR testing; and Negin Nowbarn-Nekahi for critical reading of the manuscript.
This work was supported by Public Health Service Grants DE14149-04 to J.V. and AI51946-02 to A.S.J.
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