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Cytomegalovirus Replication in Human Retinal Pigment Epithelial Cells Altered Expression of Viral Early Proteins Barbara Detrick* Jean Rhame,* Yun Wang,~f Chandrasekharam N. Nagineni,~f and John J Hooks f Purpose. Cytomegalovirus (CMV) infections are frequent complications in patients who have undergone kidney and bone marrow transplant and in patients with acquired immune deficiency syndrome. The mechanism by which CMV is activated and replicated within the retina is unknown. The authors evaluated the ability of human CMV to initiate replication in human retinal pigment epithelial (RPE) cells and compared this system with CMV replication in human fibroblasts (HEL-299, MRC-5) and human amnion epithelial (WISH) cells. Methods. Human RPE cells were obtained from donor eyes and propagated in vitro. Cells were infected, and CMV replication was evaluated in three ways: the detection of viral antigen by immunofluorescent, flow cytometry, and Western blot assays; the detection of virus-induced cytopathic effect (cpe), and the detection of infectious virus. Results. No evidence of viral replication in the epithelial (WISH) cells was found. Although CMV does not usually replicate in vitro in epithelial cells, CMV replication was detected in RPE cells. There are a number of distinct differences in CMV replication in RPE cells compared to replication in human fibroblasts. Virus-induced cpe and the production of infectious virus by RPE cells were delayed when compared to virus infection in either HEL or MRC 5 cells. At a multiplicity of infection of 0.1 and 1, cpe and infectious virus yield reached maximum levels at days 4 to 5 in fibroblasts and at days 19 to 46 in RPE cells, respectively. Nevertheless, infectious virus produced by RPE cells (10b5 TCID50/0.1 ml) significantly surpassed levels produced by HEL cells (1055 TCID50/0.1 ml). The permissive infection in RPE cells consisted of a prolonged period (5 to 6 days) of virus production in the absence of cytopathology. Virus protein expression evaluated by indirect immunofluorescence assays, Western blot analysis, and flow cytometry revealed a delay in viral protein expression in RPE cells compared to viral protein expression infibroblasts.The pattern of viral protein evaluated by flow cytometry was noticeably different in the two cell types. At the middle phase of CMV replication in RPE cells, a low percentage of cells express immediate early (IE) protein at a time when a high percentage of the cells express early (E) proteins. This IE-1 protein is a stable protein found concurrently with E protein in fibroblasts. This difference in percentage of cells expressing specific CMV proteins is transient, that is, it does not remain apparent at 100% cpe. Conclusions. Retinal pigment epithelial cells appear to demonstrate a distinct pattern of CMV infection. The low frequency of expression of IE viral protein in RPE cells, the subsequent slow replication of CMV, and the altered expression of IE viral proteins may be critical variables that impact on their relationship to viral persistence and activation within the retina. Alterations in the IE gene product may indicate the existence of positive or negative nuclear transcription factors within infected RPE cells. Invest Ophthalmol Vis Sci. 1996; 37:814-825. From the * Department of Pathology, The George Washington University Medical Center, Washington, DC, and t The Immunology & Virology Section, laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland. Presented in part at the 1993 and 1994 annual meetings of the Association for Research in Vision and Of/hllialmology, Sarasola, Florida. Submitted for publication February 10, 1995; revised November 16, 1995; accepted Deceml>er 19, 1995. Proprietary interest category: N. Reprint requests: Barbara Detrick, The George Washington University Medical Center, Ross Hall, Room 502, 2300 Eye Street NW, Washington, DC 20037. 814 .Human CMV is a herpesvirus that is a major cause of blindness in children born with congenital infections and in immunocompromised persons.1'2 During the past decade, the number of persons with compromised immune systems is rapidly increasing because of immunosuppressive therapy in cancer and transplantation and to complications of human immunodeficiency virus infection. Moreover, vision loss caused Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 CMV Replication in Human RPE Cells by CMV retinitis is a rapidly expanding problem despite the use of newer treatment modalities.3 The natural course of CMV retinitis is incompletely understood. A basic understanding of CMV replication processes within selected retinal cells may provide a rationale for alternative treatment modalities. Cytomegalovirus is a DNA virus whose replication is regulated sequentially and is dependent on viral and cellular factors.4 In permissive infection, there are three kinetic phases of gene expression: immediate early (IE), early (E), and late (L) genes. 1-4~6 Immediately after viral infection, the CMV IE genes code for IE-1 and IE-2 proteins. These genes depend on host factors for their expression, which occur in the absence of viral DNA replication. E gene expression is dependent on IE gene expression. E genes encode for enzymes required for viral DNA synthesis. After viral DNA synthesis is initiated, L genes are expressed and encode for the major part of the virion. In nonpermissive infection, these kinetic phases of viral gene expression are altered. Usually, no viral proteins are expressed or only IE proteins are detected.7 Moreover, the initial phase of IE gene activation is under the control of the major IE promoter (MIEP), whose activation is dependent on host cell transcription factors.8"' IE-1 and IE-2 proteins themselves regulate homologous and heterologous promoters.10" The restricted nature of CMV replication can thus be controlled by both viral and cellular factors. Limited cellular distribution of virus in selected tissue is a hallmark of CMV infection. Histopathologic evaluation, viral isolation, and, more recently, in situ hybridization have identified that CMV may be detected more frequently in selected tissue cell types. For example, CMV is found in ductal epithelium of salivary glands, in mucosal epithelium and submucosal endothelial cells of the intestine, in bile duct epithelium of the liver, and in alveolar and bronchial epithelium of the lungs.1213 Within the retina, CMV also has been observed within the retinal pigment epithelial cells and within the neural retina.1415 The RPE cell is a potent regulatory cell within the retina. It is known to participate in the transport of vitamin A and nutrients to the neural retina, it phagocytizes the outer segments shed by photoreceptor cells, it adsorbs light, and it provides adhesive properties for the retina."' This cell also appears to be particularly important in infectious and/or immunologic conditions within the eye.17 First, intracellular infectious agents, including viruses and parasites, can infect and replicate within this cell.18"20 Second, bacterial products, such as lipopolysaccharides and cytokines, can bind to and activate RPE cells.21'25 Third, interferon-y-activated RPE cells can express major histocompatibility class I and II molecules and the adhesion molecule, ICAM-1. Furthermore, these activated RPE cells can function as antigen-processing and antigen- 815 presenting cells within the retina.23 Finally, the infected or cytokine-activated cell can produce and release a variety of cytokines that may alter the ocular microenvironment.2b~28 The effects of CMV infection on the varied physiologic and immunologic actions of RPE cells is unknown. It is difficult to study CMV latency in humans. Currently, cell culture models of CMV replication and latency have been used to uncover clues about CMVhost interactions. Human fibroblasts often have served as the traditional cell line; however, in vivo these cells usually are not infected with CMV. Investigations using other cell models are needed to reflect more accurately this in vivo process. Because CMV replicates within human RPE cells, this pattern of virus replication may reflect replication more accurately within specialized cells and may provide novel ways to evaluate factors that control CMV gene expression and replication. In this article, we show that even though CMV usually does not replicate in epithelial cells in vitro, it can replicate within human RPE cells. However, there are a number of distinct differences in virus replication in RPE cells compared to replication in fibroblasts. Virus replication in RPE cells is atypically slow, particularly when compared to CMV replication in human fibroblasts. In addition, the permissive infection in RPE cells consists of a prolonged period (5 to 6 days) of virus production in the absence of cytopathology. Characterization of viral protein expression indicates that there is an altered pattern of CMV IE protein expression in RPE cells in comparison to fibroblasts. These distinct changes may be important findings associated with CMV infection in resident ocular cells. MATERIALS AND METHODS Cells Human RPE cells were obtained and propagated as described previously.26 When subcultured cells reached confluency, they typically were hexagonal and formed monolayers with clear intercellular boundaries characteristic of epithelial cells. Two primary cell lines of human RPE cells between passages 5 and 10 were used in this study. When different passages of these cell lines were tested for the presence of cytokeratin, 100% of the cells reacted positively with monoclonal antibodies (mAb) to cytokeratin. This reactivity supports the presence of epithelial cells. Immunoblotting analysis further confirmed the presence of cytokeratin and indicated that cytokeratin 18 (42 kDa) is the predominant form. At the time of first passage, RPE cells reacted positively with mAb developed against RPE. However, continuous culture of these cells results in the loss of reactivity with this RPE-specific mAb. As previously described, this reactivity is completely lost after the first passage of the cells.26 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 816 Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5 HEL-299 and MRC-5 are human fibroblast cell lines obtained from American Type Culture Collection (CCL137, CCL171; ATCC, Rockville, MD), and WISH cells are a human amnion epithelial cell line obtained from ATCC (CCL25). All cells were maintained in minimum essential medium supplemented with antibiotic-antimycotic (Xl) mixture (Gibco BRL, Grand Island, NY) and 2% to 5% fetal bovine serum. Human Research The tenets of the Declaration of Helsinki were followed, and informed consent was obtained from all participants. Approval was obtained from the Office of Human Subjects Research. Viruses Cytomegalovirus, AD 169 strain, was used in this study. For some studies, a clinical isolate of CMV was used. The virus was isolated from peripheral blood lymphocytes of a patient who underwent bone marrow transplantation at The George Washington University Medical Center. The virus was passaged twice in human fibroblasts. Virus stock was prepared by propagation in HEL cells. Infected cultures were harvested by freezing and thawing one time, followed by centrifugation for 20 minutes at 2000 rpm. Supernatant fluids were used as virus inoculum. Virus infectivity assays were performed on HEL cells propagated in 96-well microtiter plates. Infectivity was recorded as the induction of cytopathic effect (cpe) by serial 10-fold dilutions of the sample. Antibodies Murine monoclonal antibodies directed against human CMV immediate IE, E, and L antigen were obtained (Accurate Chemical 8c Scientific, Westbury, NY). The MAS127 clone 13 is an immunoglobulin (IgG) 1 antibody that reacts with IE nonstructural antigen of 72 kDa (IE-1). The MAS 550 clone 2A2 is an IgGl antibody that reacts with "early" antigen present 16 hours after infection. The MAS 552 clone SL-20 is an IgG3 antibody that reacts with "late" antigen present 72 hours after infection. Monoclonal antibodies (IgGl and IgG3) were used as isotypic controls. Indirect Immunofluorescent Assay Retinal pigment epithelial, HEL, and WISH cells were propagated on chamber slides. Cells were incubated with CMV at an input multiplicity of 0.5 to 1 for 2 hours, washed three times in PBS, re-fed with maintenance media, and incubated at 37°C. Cells were then incubated at 37°C and at various times after infection, removed, and processed for immunofluorescent assays. Slides containing cells were air dried and fixed in acetone for 4 minutes and washed three times in PBS. Primary antibodies were applied for 30 minutes in a humidified chamber. Slides were washed three times in PBS, followed by incubation with fluoresceinconjugated horse anti-mouse IgG for 30 minutes. Slides were washed three times again, mounted, and viewed under a fluorescent microscope. For indirect immunofluorescence studies, slides were reviewed in a coded fashion by three investigators. The following samples were evaluated in the immunofluorescent assay: uninfected cells incubated with media, irrelevant mAb, or virus-specific antibodies; or infected cells incubated with media, irrelevant mAb, and serial 2-fold dilutions of the virus-specific antibodies. These cultures were then incubated with the second fluorescein isothiocyanate conjugate-labeled anti-mouse Ig antibody. The irrelevant mAbs were the same isotypes as the positive anti-virus antibodies and were used at the same concentration as the sample tested. Flow Cytometric Analysis Uninfected and CMV-infected RPE and HEL cells were removed from plastic dishes with trypsin-versine. Cell suspensions were fixed in 100% methanol for 10 minutes at 4°C. After fixation, cells were washed twice with PBS, and the cell count was adjusted to 10,000 cells///l. Primary incubations of 100 fj\ of cells, with appropriate dilutions of isotypic controls and monoclonal antibodies, were for 30 minutes at 4°C. After washing with PBS, fluorescein isothiocyanate goat F(ab)2 anti-mouse antibody was added, and the cells were incubated for an additional 30 minutes at 4°C. Cells were again washed twice with PBS, then fixed with 1% paraformaldehyde. All samples were held at 4°C until analyzed. Analysis was performed on a Coulter (Hiahleah, FL) Profile II flow cytometer equipped with a 488 argon laser. Forward and side angle light scatter were used for gating purposes, and 10,000 cells were collected. Isotypic controls, establishing the nonspecific binding region, were followed by the associated monoclonal antibodies to determine percent positive fluorescence. Data presented are representative of at least three separate experiments. Western Blot Analysis Uninfected and CMV-infected human RPE cells were lysed by sonication in extraction buffer (50 raM Tris, pH 8.5; 4 mM EDTA; 2 mM phenylmethylsulfonyl fluoride; 1% [wt/vol] deoxycholate; 1% [vol/vol] Triton X-100). Cell lysates were clarified by centrifugation at 10,000 rpm for 10 minutes. Samples were adjusted to a concentration of 20 /^g/40 [i\. The supernatant fraction was subjected to sodium dodecyl sulfate-10% polyacrylamide electrophoresis and transferred to nitrocellulose membranes. After a 6-hour incubation of the membranes with either anti-CMV IE, anti-CMV E, or anti-CMV L protein antibodies, they were washed and further incubated for 2 hours with a 1:100 dilution Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 817 CMV Replication in Human RPE Cells FIGURE 1. Cytomegalovirus-induced cytopathology in human fibroblast cells (HEL). (A) Normal, uninfected HEL cells. (B) Appearance of HEL cells 2 days after infection with CMV at a multiplicity of infection of 0.1. of goat anti-mouse IgG coupled to horseradish peroxidase. The color was developed with 4-chloro-l-naphthol and H^O-z as substrates. Electron Microscopy The cell culture medium was decanted, and the cells were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4) at 4°C. After 20 minutes, the cells were scraped gently with a rubber policeman, poured into a polypropylene test tube, and centrifuged into a pellet. After a 2-hour incubation at 4°C, the fixative was removed, and the pellet was stored in 0.13 M sodium phosphate buffer at 4°C. The cells were fixed in phosphate-buffered osmium tetroxide, dehydrated in alcohol, and embedded in Spurr's medium. Ultra-thin sections stained with uranyl acetate and lead citrate were examined with a Zeiss (Jena, Germany) electron microscope. RESULTS Comparison of the Development of VirusInduced Cytopathology in Human Retinal Pigment Epithelium, Fibroblasts, and Epithelial Cells Confluent monolayers of RPE, HEL-299, MRG-5, and WISH cells were incubated with CMV at an input multiplicity of infection of 0.1 and 1. Typical CMV-cpe consisting of ballooned swollen cells was noted in fibroblasts (HEL & MRC-5) within 24 hours after inoculation and encompassed 100% of the cells by days 4 to 5 (Fig. 1). In contrast, CMV did not alter the appearance of WISH cells. Cytomegalovirus-induced cpe was noted in RPE cells (Fig. 2); however, the timing was markedly different than that observed in the fibroblast cell lines. Cytopathic effect wasfirstdetected in small foci (<1% of the culture) at days 8 to 10, FIGURE 2. Cytomegalovirus-induced cytopathology in human retinal pigment epithelial (RPE) cells. (A) Normal, uninfected RPE cells. (B) Appearance of RPE cells 30 days after infection with CMV at a multiplicity of infection of 0.1. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5 818 20- Days FIGURE 3. Development of cytopathic effect (cpe) in cytomegalovirus (CMV)-infected cells. Human retinal pigment epithelial cells and human fibroblasts (HEL, MRC-5) were incubated with CMV at a multiplicity of infection of 1. After a 2-hour incubation period, inoculum was removed, and cells were washed three times and then re-fed maintenance media. Cells were evaluated daily for the development of cpe. and 100% of the cells were not involved until approximately day 22 after inoculation (Fig. 3). These studies indicate that CMV, which does not usually replicate and induce cpe in vitro in epithelial cells, can do so in human RPE cells. It is possible that a virus can induce cytopathology in the absence of a productive infection. To investigate this possibility, we next evaluated the cultures for the presence of infectious virus. Replication of Cytomegalovirus in Human Retinal Pigment Epithelial Cells Monolayers of RPE, HEL, and MRC-5 cells were incubated with CMV at an input multiplicity of 1. After a 2hour virus adsorption period, cells were washed three times, re-fed, and incubated at 37°C. At varying times, supernatant fluids were removed and assayed for virus infectivity. The remaining cells were resuspended in the same volume of media and assayed for cell-associated infectious virus. The data presented in Figure 4 show the development of infectious virus in the supernatant fluid of CMV-infected cells. Production and release of infectious virus into the supernatant fluid occurs rapidly in human fibroblasts (HEL and MRC5 cells) (Fig. 4). At day 2, CMV was detected at a concentration of 10' 5 TCID50/0.1 ml. Maximum virus titers were detected at day 4 in both fibroblast cell lines. In contrast, in RPE cells, CMV was detected at day 7 and reached a maximum at day 19 (1065 TCID50/O.I ml). The data that describe the development of cpe (Fig. 3) and the data that depict the development of infectious virus (Fig. 4) are derived from the same experiments. It is of interest to point out that high levels of infectious virus are present in the supernatant fluid of infected RPE cultures at a time (days 7 to 12) when less than 1% of the cells demonstrate a cpe. The studies shown in Figure 4 indicate that the CMV replication cycle in RPE cells is delayed in comparison to the virus replication cycle in human fibroblasts. This was noted also at different multiplicities of infection (MOI). At an MOI of 1, virus-infected RPE cells yielded maximum virus titers (10(l-5) at day 19, whereas virus-infected HEL cells yielded maximum virus titers (1050) at day 4. At a MOI of 0.1, virusinfected RPE cells yielded maximal virus titer (10b5) at day 46, whereas virus-infected HEL cells yielded maximum virus titer (1055) at day 5. These studies indicate that CMV replication in human RPE cells is delayed when compared to the time course of CMV replication in fibroblast cells. Nevertheless, the amount of infectious virus produced by RPE cells significantly exceeds the maximum levels attained in fibroblast (HEL or MRC-5) cultures. This was observed when we compared peak levels of virus produced in supernatant fluids or when we compared peak levels of virus detected in a combination of supernatant fluids and cell-associated virus (cell-associated [c] and supernatant fluid [s] virus in HEL cells versus RPE cells, P = 0.001; c + s virus in MRC-5 cells versus RPE cells, P < 0.0001; c + s virus HEL cells versus c + s Days FIGURE 4. Cytomegalovirus (CMV) replication in human fibroblasts (HEL, MRC-5) and retinal pigment epithelial cells. Cells were incubated with CMV at an input multiplicity of infection of 1. After a 2-hour incubation period, inoculum was removed, and cells were washed three times and then re-fed maintenance media. At varying times between days 1 and 22, supernatant fluids were removed and assayed for infectious virus on HEL cells. Results are expressed as the mean ± standard error of the mean for experiments performed in triplicate. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 CMV Replication in Human RPE Cells 819 virus MRC cells, P = 0.07; paired Kests). It should be noted that when cell cultures were infected, all of the cultures contained the same number of cells inoculated with the same amount of virus (MOI 1). However, optimal virus yields were obtained in 4 days in fibroblast cultures and in 19 days in RPE cultures. Therefore, the ability of CMV to reach higher maximal virus yield in RPE cells than in fibroblasts may be explained, in part, by the additional time and RPE cell multiplication. When a clinical isolate of CMV was used to infect RPE and HEL cells, a similar pattern of virus-induced cpe and replication was noted. The clinical isolate induced cpe in fibroblasts within 2 days, whereas cpe was not noted in RPE cells until 15 days. Passage of this isolate in RPE cells did not alter the delayed pattern of virus replication described above in this section. Because herpesvirus replication is frequendy cell associated, we compared the level of infectious virus produced in the supernatant fluid with the level of virus obtained from cell-associated samples. Cytomegalovirus-infected RPE, HEL, and MRC-5 cells assayed in Figure 3 also were analyzed for cell-associated virus. Data obtained for the RPE cells is shown in Figure 5. The amount of infectious virus was consistently slightly higher in the cell-associated samples. Similarly, the virus yield in the cell-associated samples for HEL and MRC-5 cells was higher than that observed in the supernatant fluids (data not shown). Electron microscopic evaluation confirmed the presence of CMV infection in RPE cells. Cytomegalovirus-infected RPE cells revealed typical herpesvirus particles within the nucleus and cytoplasm of the RPE cells. Virus particles were observed budding from the nuclear and cell membranes (data not shown). Analysis of Cytomegalovirus Proteins by Western Blot Assays Uninfected RPE cells and RPE cells infected with CMV at a MOI of 0.1 were harvested at days 10, 15, and 22 after inoculation and were analyzed by Western blot. Monoclonal antibodies reactive to CMV proteins IE1, E, or L were used. As seen in Figure 6, reactivity with IE-1, E, and L proteins was detected at days 15 and 22. A weak band of reactivity was observed at day 10 for the E protein. Infected RPE cells did not react with an unrelated monoclonal antibody, OX-8. In addition, uninfected RPE cells did not react with the monoclonal antibodies for CMV proteins IE-1, E, or L. At this multiplicity of infection (0.1), the kinetics of detectable virus by infectivity assays and detection of viral proteins by Western blot assays were similar. Analysis of the Sequential Expression of Cytomegalovirus Proteins in Retinal Pigment Epithelium, Fibroblasts, and Epithelial Cells by Immunofluorescent Assays Retinal pigment epithelial, HEL, and WISH cells propagated on chamber slides were incubated with CMV Days FIGURE 5. Cytomegalovirus (CMV) replication in human retinal pigment epithelial cells. Cells were incubated with CMV at an input multiplicity of infection of 1. After a 2-hour incubation period, inoculum was removed, and cells were washed three times and then re-fed maintenance media. At varying times between days 1 and 22, CMV-infected RPE culture lysates or supernatants were assayed for the production of cell-associated or extracellular virus. Results are expressed as the mean ± standard error of the mean for two separate experiments, each of which was performed in triplicate. at an input multiplicity of 1. At varying days after inoculation (days 1 to 4), slides were fixed in acetone and incubated with monoclonal antibodies reactive to CMV IE-1, E, or L proteins and to an unrelated epitope, OX-8. Data obtained are summarized in Table 1. In CMV-infected HEL cells, the nuclear staining for IE-1 protein was detected within 5 hours. This reactivity was observed at days 1 to 4 in more than 90% of cells. Nuclear staining for E protein was first noted at 24 hours. This reactivity persisted and was observed on days 2, 3, and 4 in more than 90% of the cells. Diffuse cytoplasmic staining for L protein was first noted at 48 hours. In CMV-infected RPE cells, IE-1 protein reactivity was weak at day 1. At 48 hours, reactivity was observed clearly. Although clear, bright nuclear fluorescent staining for IE-1 protein could be detected in the RPE cells, it was observed in less than 1% of the cell population. This is in sharp contrast to the staining pattern observed in HEL cells, in which more than 90% of the cells were reactive within 48 hours. E protein was detected at 48 hours, and weak reactivity for L protein was detected at 72 hours. In contrast to the reactivity noted in HEL and RPE cells, CMV proteins IE-1, E, and L were not detected in CMV-inoculated WISH cells. The immunofluorescent staining data correlate with the development of cpe and infectious virus noted in HEL, RPE, and WISH cells. Retinal pigment epithelial cells evaluated after day Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5 820 TABLE l. Comparison of the Expression of CMV Proteins on HEL, Human RPE, and WISH Cells IE Protein Incubation (days) HEL RPE L Protein E Protein WISH HEL RPE WISH HEL RPE WISH 1 Reactivity >90 0 Reactivity >90 50 50 >90 >90 >60 100 100 100 Reactivity Reactivity <1 — = negative; ± weakly positive; + = positive; ++ = positive with increased intensity; % = percentage of cells reactive; HEL = human fibroblast cell line; REP = retinal pigment epithelium; WISH = human amnion epithelial cell line; CMV = cytomegalovirus. 4 demonstrated an increase in the expression of IE, E, and L proteins. When cytopathology was seen in 100% of the cells, 90% were positive for IE protein and 100% were positive for E and L proteins. Analysis of Cytomegalovirus Protein Expression by Flow Cytometry To quantitate accurately the percentage of cells staining and the intensity of the staining with anti-CMV antibodies, these cultures were analyzed by flow cytometry. Seventy-two hours after CMV inoculation (MOI of 0.1), HEL cells were harvested and evaluated by flow cytometry. As is seen in Figure 7, 89.3% of the cells were reactive for IE-1 protein, and 98.3% of the cells were reactive for E protein. A similar pattern of antigen expression has been described for IE-1 and E proteins in CMV-infected fibroblast cells. This study underscores the usefulness of flow cytometry for quantitating the number of cells expressing CMV proteins. Four, 8, 26, and 30 days after CMV inoculation (MOI of 0.1), RPE cells were harvested and evaluated by flow cytometry (Fig. 8). Flow cytometric analysis revealed no reactivity in CMV samples harvested 4 or 8 days after inoculation. In contrast, samples harvested on day 26 were 19% reactive for IE-1, 84% reactive for E, and 5% reactive for L proteins. In a separate experiment, CMV protein expression was monitored 30 days after inoculation of RPE cells. Again, only 17.8% of the cells were reactive for IE-1, whereas 89.6% were reactive for E and 33.9% were reactive for L proteins. Because of cell integrity requirements of flow cytometry, it was not possible to evaluate the RPE cells beyond this time point or when cpe was 100%. Overall, these studies underscore a striking difference in the percentage of cells staining for the IE-1 protein. By flow cytometric analysis, we identified an interesting pattern of viral gene expression in the mid- dle of the CMV replication cycle. At this phase of CMV replication in RPE cells, a low percentage of cells express IE proteins at a time when a high percentage of cells in the population express E proteins. This CMV IE-1 protein is a stable protein found concurrently with E protein expression in HEL cells. This difference in the percentage of cells expressing CMV IE proteins in RPE cells is transient, that is, it does not remain apparent at the time of 100% cpe (evaluation by immunofluorescent assays). This altered pattern of viral protein expression may be reminiscent of recent studies29 identifying a transient expression of CMV IE proteins in CMV-infected macrophages. DISCUSSION This study demonstrates that CMV can infect and replicate in human RPE cells. The virus induces a typical CMV cytopathology in cultured RPE cells and HEL cells, but not in another epithelial cell, WISH. Furthermore, this article describes an unusual pattern of viral infection, that is, the virus replicates very slowly in RPE cells. Although CMV induces cytopathology and cell death in RPE cells, there is a prolonged period (days 6 to 12) of virus production in the absence of cytopathology. This pattern is strikingly different from CMV infection in fibroblasts. Moreover, there appears to be a unique pattern of viral proteins expressed at the mid-point during CMV infection in RPE cells. This shift is not seen in HEL cells. For example, at a time when more than 89% of HEL-infected cells expressed both IE and E proteins, RPE cells expressed a disproportionate level of these two proteins. Specifically, RPE cells expressed 20% IE and 90% E proteins. Finally, of the three methods used to monitor this virus infection, flow cytometric analysis allowed us to quantify the expression of CMV-induced proteins. In vivo Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 821 CMV Replication in Human RPE Cells CMV infection frequently is associated with epithelial cells, not fibroblasts. The kinetics of CMV replication in RPE cells may not be the exception but rather the typical mode of replication in specialized cells. Cytomegalovirus infections are characterized by a marked restriction in susceptible cells.1 In vitro studies usually have relied on the efficient replication of CMV in human fibroblast cells. However, a few reports have identified low levels of CMV replication in cultured o in > > CO cj 106 80 - 49.5 - 1 CO 1 CM CM > CO T» 0) 1 o o C > > > C '£ 2 S 2 "c o o o >. CO • in >» CO •D • > > o O CM CM CO •a • > 2 o 32.5 27.5 18.5 control 106 80 - 49.5 - CMV - IE - protein 32.5 27.5 18.5 CMV - E - protein CMV - L - protein FIGURE 6. Western blot analysis of cytomegalovirus (CMV) proteins in uninfected and CMV-infected retinal pigment epithelial cells. Cells were infected with CMV at a multiplicity of infection of 0.1. Uninfected and CMV-infected RPE cells were harvested as described in Materials and Methods. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the samples were transferred to nitrocellulose membranes and incubated with monoclonal antibodies, which react with immediate early (IE)-l, early (E), or late (L) viral proteins. Protein molecular weight markers (in thousands) are shown on the left. epithelial cells, endothelial cells, and peripheral blood mononuclear cell lines. In these cell types, the efficiency of CMV replication appeared to depend on the maturity level and ploidy of the cell.30"*3 For example, in the monocyte-macrophage cell system, CMV can infect monocyte progenitors in bone marrow cells. The CMV genome can exist within these cells for prolonged periods with litde or no viral gene expression. When the cells reach a certain stage of differentiation, the IE, E, and L genes are expressed sequentially.7tM Alternatively, CMV replication in a differentiated monocyte cell line, THP-1, is associated with changes in viral enhancer-binding proteins and IE-1 gene expression, which occur with initiation of differentiation.32-35 The fact that CMV replicates in RPE cells indicates that these cells are at a stage of differentiation to be permissive. The shift from a low level of permissive cells early in the infection to 100% permissive cells may reflect alterations in cell cycle or level of cellular differentiation. Preliminary studies in our laboratory indicate that when RPE cells are maintained in serum-free conditions, the cells morphologically appear more differentiated. Cytomegalovirus infection of these cells occurs more rapidly than in RPE cells maintained in serum (data not shown). Studies are under way to evaluate the conditions that allow these serum-free RPE cells to become more permissive to CMV infection. Of particular interest in this study was the observation that the slow replication of CMV in RPE cells was associated with a low percentage of the cells displaying viral proteins during the first 10 days after infection. There are at least two possible explanations for the low number of RPE cells expressing viral proteins, in particular, the IE proteins. First, virus attachment and penetration may be inhibited; second, IE gene expression may be altered by viral or RPE cellular transcription proteins, or both.1 The initiation of CMV infection is characterized by attachment of the virus to the cell membrane and penetration by the virion into die cell. Three viral proteins are associated with this cellular attachment.1 A One such protein, B2 microglobulin, is found on the virion surface, which may bind to MHC proteins on the cell surface. In addition, viral glycoprotein H(gH) and glycoprotein b (gB) bind to 90 kDa and 30 kDa cellular proteins, respectively. The distribution or concentration of these cellular receptors on RPE cells may be insufficient to support CMV infection. This may result in a low number of RPE cells expressing IE protein. In fact, it has been reported that the MHC molecules are either not expressed or are expressed in low concentration on nonactivated RPE cells.36 Alternatively, the low number of CMV-infected RPE cells during the first 10 days may be associated with viral and/or cellular control of IE gene expression. It is known that IE gene expression is influenced Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 822 Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5 control antibody (1.4%) No. of Cells Log Fluorescence Intensity FIGURE 7. Flow cytometric analysis of cytomegalovirus (CMV) proteins in HEL cells. Flow cytometric analysis of CMV-infected HEL cells (3 days after inoculation), demonstrating 1.4% positivity for the isotypic control, 89.3% for the immediate early (IE) CMV protein, and 99.3% positive for the early (E) CMV protein. by cellular factors, such as NFkB.4810 12 IE genes are controlled by the major IE promoter (MIEP) whose activation depends on such host cell factors as cellular transcription proteins. A number of investigators37 have shown that HCMV MIEP expression is different in nonpermissive and permissive cells. Nonpermissive cells may either lack positive transcriptional regulatory factors or possess a repressor protein that acts on the MIEP.38'39 On the other hand, permissive cells may contain these positive transcriptional regulatory factors that support virus replication. Additional studies are required to identify whether cellular regulatory proteins are involved in the transient expression of IE gene expression. In addition to our description of alterations in the early phase of CMV-infected RPE cells, flow cytometry allowed us to identify a unique pattern of virus gene expression in the middle of the CMV replication cycle. This phase of CMV replication in RPE cells is characterized by a low percentage of cells expressing IE proteins at a time when a high percentage of the cells in the population express E proteins. The IE-1, a 72-kDa protein, is a stable protein found concurrently with E protein expression in HEL cells. Whether viral factors, such as IE-2, another immediate early protein, or cellular regulatory proteins are active in downregulating IE protein expression in RPE cells remains to be determined.4041 Nevertheless, the low expression of IE-1 proteins late in RPE cell infection may be important in viral persistence. Recendy, Fish and associates29 have demonstrated that the growth kinetics of human CMV are altered in monocyte-derived macrophages. They described the productive nonlytic growth of CMV in these cells, in which the replication cycle is delayed No. of Cells Log Fluorescence Intensity FIGURE 8. Flow cytometric analysis of cytomegalovirus (CMV) proteins in retinal pigment epithelial (RPE) cells. Flow cytometric analysis of CMV-infected RPE cells (30 days after inoculation), demonstrating 1.6% positivity for the isotypic control, 17.8% for the immediate early (IE) CMV protein, 89.6% for the early (E) CMV protein, and 33.9% for the late (L) CMV protein. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933416/ on 06/19/2017 CMV Replication in Human RPE Cells relative to replication of the virus in human fibroblasts. These investigators also showed that IE protein expression appears early (day 3), disappears between days 3 and 7, and reappears with L protein at day 7. This pattern of viral protein expression appears reminiscent of CMV infection in RPE cells. Two general tenets of viral persistence are downregulation of viral gene expression and failure of the immune system to detect and clear virus-infected cells.42 These principles are true for infections, including CMV infections. Our observation of a low expression of CMV IE proteins in RPE cells may contribute to viral persistence within the retina by evading immune elimination. Cell-mediated immune responses are important in controlling CMV infections.43 In fact, in the murine system cytotoxic T cells (CTLs), CD8+ T cells, are instrumental in host defense against CMV infections.43 Adoptive transfer of CD8+ T cells prevents death in the mouse CMV immunodeficiency model. The majority of the CTLs recognize immediate early products and not late viral gene products. In fact, as many as 50% of the CTLs recognize IE-1 gene products. 44 ' 45 Additional studies with a vaccinia virus expressing CMV IE-1 proteins demonstrated the importance of these proteins in CMV-host cell interactions. 46 Inoculation of mice with the vaccinia virus expressing only CMV IE-1 proteins protected mice from a lethal challenge of CMV. In humans, CTLs specific for CMV IE proteins also have been described and shown to play a critical role in controlling CMV infections.47"49 The studies reported here show that during the first 10 days after infection, there is a low level of expression of all viral proteins in CMV-infected RPE cells. When virus replication increases, low levels of IE protein are observed at a time when greater amounts of E and L proteins are expressed. Because the majority of CTLs generated in CMV infections can be directed against IE proteins, this alteration in the eye may allow CMVinfected RPE cells to evade immune destruction. 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