Zika Virus Infection of Human Iris Pigment Epithelial Cells.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells.

Human cytomegalovirus infects multiple cell types, including fibroblasts and epithelial cells. It penetrates fibroblasts by fusion at the cell surface but is endocytosed into epithelial cells. In this report, we demonstrate by electron microscopy that the virus uses two different routes to enter retinal pigmented epithelial cells, depending on the cell type in which the infecting virus was produced. Virus produced in epithelial cells preferentially fuses with the plasma membrane, whereas fibroblast-derived virus mostly enters by receptor-mediated endocytosis. Treatment of epithelial cells with agents that block endosome acidification inhibited infection by virus produced in fibroblasts but had only a modest effect on infection by virus from epithelial cells. Epithelial cell-generated virions had higher intrinsic "fusion-from-without" activity than fibroblast-generated particles, and the two virus preparations triggered different cellular signaling responses, as evidenced by markedly different transcriptional profiles. We propose that the cell type in which a human cytomegalovirus particle is produced likely influences its subsequent spread and its contribution to pathogenesis.

Study of Usutu virus neuropathogenicity in mice and human cellular models.

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.

In vitro replication of varicella-zoster virus in human retinal pigment epithelial cells.

Here we describe for the first time the productive in vitro infection of human retinal pigment epithelial cells by varicella-zoster virus (VZV), resulting in a typical cytopathic effect (CPE) that is characterized by enlarged cells with increased granularity. Depending on the CPE dissemination, high titers of up to 1.6 x 10(6) PFU of cell-free and cryostable VZV/ml can be recovered.

Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector.

PURPOSE: To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye. METHODS: Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65). The cell specificity of this novel hybrid promoter was tested in human RPE (ARPE-19), human glioblastoma, rat C6 glioma, mouse hippocampal neurons, and human embryonic kidney cell lines. Expression of all cell types in normoxia, and following 40 h hypoxia, was analyzed by dual luciferase assay. After confirmation of its tissue-specificity and hypoxia-inducibility, the hybrid promoter construct was integrated into a replication-deficient AAV delivery system and tested for cell-selectivity and hypoxia-inducible green fluorescent protein (GFP) reporter expression. RESULTS: The HRSE-6xHRE-Rpe65 promoter was highly selective for RPE cells, strongly induced in hypoxia, and similar in activation strength to the cytomegalovirus (CMV) promoter. The AAV.HRSE.6xHRE.Rpe65 vector induced robust GFP expression in hypoxic ARPE-19 cells, but elicited no GFP expression in hypoxia in other cell types or in normoxic ARPE-19 cells. CONCLUSIONS: The hypoxia-regulated, aerobically-silenced RPE-targeting vector forms a platform for focal autoregulated delivery of antiangiogenic agents in hypoxic regions of the RPE. Such autoinitiated therapy provides a means for early intervention in choroidal neovascularization, when it is most sensitive to inhibition.

Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway.

Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.

HTLV-1 infection of human retinal pigment epithelial cells and inhibition of viral infection by an antibody to ICAM-1.

PURPOSE: To examine whether human T-cell leukemia virus type 1 (HTLV-1) could infect a human retinal pigment epithelial (RPE) cell line, ARPE-19, in vitro and to investigate its regulation. METHODS: A coculture system with ARPE-19 and irradiated cells of an HTLV-1-producing T-cell line, MT2 was used to determine the permissivity of RPE to HTLV-1 infection in vitro. The susceptibility to HTLV-1 was assessed by detection of viral DNA using the polymerase chain reaction (PCR), viral mRNA transcripts with reverse transcription PCR (RT-PCR) and viral antigen by immunofluorescence staining. An HTLV-1 Tax-activated HTLV-LTR-luciferase reporter assay was developed to measure viral infection quantitatively. The ICAM-1 expression on cocultured ARPE-19 cells was detected by flow cytometry and an ICAM-1-neutralizing antibody was used to test ICAM-1's role in the HTLV-1 infection of ARPE-19 cells. The regulation of HTLV-1 infection was investigated by culturing ARPE-19 cells with proinflammatory cytokines. RESULTS: HTLV-1 infected ARPE-19 cells in vitro. The infection correlated with elevated expression of intercellular adhesion molecule (ICAM)-1 on the surface of ARPE-19 cells. ICAM-1-neutralizing antibody dramatically inhibited viral infection. Furthermore, proinflammatory cytokines dramatically suppressed HTLV-1 viral infection. CONCLUSIONS: The tropism of HTLV-1 to retinal pigment epithelium could provide an explanation for the pathogenesis of HTLV-1-related ophthalmic diseases. A better understanding of specific roles of proinflammatory cytokines in the development of ophthalmic diseases may be beneficial for treatment.

West Nile virus infection induces interferon signalling in human retinal pigment epithelial cells.

PURPOSE: In addition to neuroinvasive disease, West Nile virus (WNV) infection is frequently associated with self-limiting chorioretinitis and vitritis. However, the mechanisms of ophthalmic WNV infection are rarely investigated, in part because of the lack of reliable in vitro models. The authors therefore established the first model of ocular WNV infection and investigated interaction of WNV with IFN signal-transduction mechanisms. METHODS: Human retinal pigment epithelial (RPE) cells were infected with WNV strain NY385-99 at a multiplicity of infection of 5. Virus replication was evaluated by virus titers at different times after infection. The susceptibility of RPE cells to WNV infection was confirmed by transmission electron microscopy. IFN-beta expression was assessed by quantitative real-time PCR and by measurements of antiviral activity in cell culture supernatants. IFN signaling was evaluated by phosphorylation of transducer and activator of transcription 1 and 2 (STAT1/2) proteins, with immunoblot analysis. RESULTS: RPE cells appeared to be highly sensitive to WNV infection. Maximum viral titers were found 24 hours after infection, followed by a continuous decline during the course of infection. WNV infection of RPE cells was followed by increased IFN-beta expression associated with IFN signaling and subsequent inhibition of WNV replication. CONCLUSIONS: In this study, the first cell culture model of ophthalmic WNV infection was developed and characterized in RPE cells, and the molecular mechanisms of WNV infection were studied. The data suggest that WNV induces a general antiviral state in RPE cells. This general antiviral state correlates with WNV-induced IFN signaling in retinal cells.

Recombinant AAV-transduced iris pigment epithelial cell transplantation may transfer vector to native RPE but suppress systemic dissemination.

PURPOSE: To determine whether adenoassociated virus (AAV) vectors transduced into iris pigment epithelial (IPE) cells and transplanted into the subretinal space of rats will transfer the AAV genome to the host cells and whether the vectors are disseminated systemically. METHODS: Recombinant (r)AAV was transduced into rat IPE cells and transplanted into the subretinal space of rats. For the control, rAAVs alone were injected subretinally. The transplanted IPE cells were detected by LacZ staining. Immunohistochemistry, electron microscopy, electroretinography, and fluorescein-dextran angiography were performed. DNA was extracted from various organs and blood and examined for the AAV genome by polymerase chain reaction. RESULTS: No toxicity from rAAV transduction was observed in vitro. LacZ was expressed in the transplanted cells 1 and 2 weeks after transplantation. At 4 and 12 weeks, fewer transplanted cells were detected than at 1 week, and LacZ expression was occasionally detected at the level of host retinal pigment epithelial (RPE) cells. Expression was also detected in ciliary body epithelial cells. The electroretinograms and fluorescein-dextran angiography were only mildly altered. Significantly lower levels of AAV genome were detected in the organs and blood of rats receiving rAAV-IPE cell transplants than with direct intravenous injection of AAV vectors. CONCLUSIONS: AAV-mediated LacZ was expressed in the transplanted cells after subretinal transplantation, and the transplanted IPE cells may transfer the rAAV to host tissues, such as RPE cells, long after the transplantation. This method of gene delivery did not lead to systemic dissemination of the vectors.

Human cytomegalovirus temporally regulated gene expression in differentiated, immortalized retinal pigment epithelial cells.

BACKGROUND: Human cytomegalovirus (HCMV) replication in epithelial cells is crucial for its pathogenesis. To date, HCMV gene expression has been primarily studied in human foreskin fibroblasts (HFFs), although their importance for HCMV pathogenesis remains unclear. Primary retinal pigment epithelial (RPE) cells are permissive for HCMV. OBJECTIVES: Our objectives were to determine the production of alternatively processed HCMV major immediate-early (MIE) and UL37 RNAs and their essential products in infected, terminally differentiated immortalized RPE (hTERT-RPE) cells. STUDY DESIGN: hTERT-RPE cells were studied because of their notable similarities with primary RPE cells, and because they overcome key limitations of primary cells. hTERT-RPE cells were terminally differentiated in vitro and infected with HCMV. Total RNA or cell proteins were analyzed at various times post-infection. RESULTS: We show for the first time that HCMV-infected, differentiated hTERT-RPE cells produce IE1, IE2, UL37 exon 1 (UL37x1) and UL37 alternatively spliced RNAs, albeit with abundances and kinetics distinct from those observed in HCMV-infected HFFs. IE1-72 was produced in HCMV-infected, differentiated hTERT-RPEs within 24h post-infection (hpi); whereas, IE2-86 and pUL37x1 were produced within 72 hpi. IE2-86 was detected after IE1-72 even though its transcript appeared first. Early/late (pp65) and late (pp28) proteins were produced within 96-120 hpi. CONCLUSIONS: The temporal cascade of HCMV gene expression was observed in infected, differentiated hTERT-RPE cells. Moreover, HCMV IE RNAs are alternatively and accurately processed in differentiated hTERT-RPE cells. However, the delayed temporal expression suggests further regulation of HCMV gene expression at post-transcriptional/translational levels in differentiated hTERT-RPE cells.

Nicotine treatment alters NF-kappaB expression in human cytomegalovirus-infected ARPE-19 cells.

PURPOSE: To test if nicotine counteracts the dampening effect of human cytomegalovirus (HCMV) infection of NF-kappaB in retinal pigment epithelial (RPE) cells, thereby increasing the permissiveness of RPE cells for HCMV replication. METHODS: Human ARPE-19 cells were transfected with NF-kappaB luciferase DNA, inoculated with HCMV at 24 hr post-transfection, and maintained in the absence or presence of a physiologic dose of nicotine at 1 hr prior to HCMV inoculation. RESULTS: Whereas HCMV-infected ARPE-19 cells without nicotine treatment showed a dramatic decrease in NF-kappaB levels, nicotine treatment reduced this decrease but did not abolish it completely. Nicotine treatment of uninfected ARPE-19 cells had no effect on baseline NF-kappaB levels. CONCLUSIONS: Treatment of HCMV-infected ARPE-19 cells with nicotine at a physiologic dose dampened the downregulation of NF-kappaB observed in HCMV-infected ARPE-19 cells without nicotine treatment. We conclude that nicotine can serve as a cofactor to stimulate productive, lytic replication of HCMV.

Human cytomegalovirus retinitis: pathogenicity, immune evasion and persistence.

Human cytomegalovirus (HCMV) retinitis frequently occurs in severely naturally and iatrogenically immunocompromised patients. It has been shown that the immune-privileged retinal pigment epithelium (RPE) is a major site of persistent HCMV. Recently, evidence has accumulated to show that HCMV immediate early (IE) gene expression in RPE cells deviates ocular antiviral inflammation via FasL. Moreover, unlike in other cell types, the HCMV major IE1/2 enhancer promoter (MIEP) resists activation by proinflammatory stimuli mediated by the transcription factor NF-kappaB. However, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) found at elevated levels in transplant recipients and AIDS patients with retinitis sensitize RPE cells and other retinal cells to FasL-mediated apoptosis, thus contributing to retina destruction and necrosis rather than inflammation. These specific features of RPE cells in conjunction with deregulated immune responses of immunocompromised patients seem to contribute to virus persistence and pathogenesis within the immune-privileged ocular retina.

Increased replication of human cytomegalovirus in retinal pigment epithelial cells by valproic acid depends on histone deacetylase inhibition.

PURPOSE: Human cytomegalovirus (HCMV) replication depends on different cellular pathways, including histone acetylation and extracellular-signal regulated kinases 1 and 2 (Erk 1/2). In the present study, the influence of therapeutic valproic acid (VPA) concentrations was investigated on HCMV replication in retinal pigment epithelial (RPE) cells. METHODS: HCMV antigen expression and replication were detected by immunostaining, real-time RT-PCR, and determination of virus titers. Histone acetylation and Erk 1/2 phosphorylation were detected by Western blot. RESULTS: Pretreatment with VPA < or =1 mM enhanced HCMV antigen expression and replication by up to ninefold. In addition to histone deacetylase (HDAC) inhibition, VPA stimulated Erk 1/2 phosphorylation in RPE cells. Investigation of six VPA derivatives revealed that S-2-pentyl-4-pentynoic acid was the only derivative that induced histone hyperacetylation, indicating HDAC inhibition, in the observed concentrations < or =1 mM and that increased HCMV antigen expression. Other derivatives did not enhance HCMV replication in the tested concentrations, although some were found to induce Erk 1/2 phosphorylation. The mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 inhibited VPA-induced Erk 1/2 phosphorylation but did not affect VPA-induced increased HCMV replication. In addition, the structurally nonrelated HDACI trichostatin A enhanced HCMV replication but did not affect Erk 1/2 phosphorylation in RPE cells. CONCLUSIONS: The data demonstrate that VPA stimulates HCMV replication by HDAC inhibition independent of Erk 1/2 phosphorylation in therapeutic concentrations in RPE cells. Therefore, patients at risk of HCMV retinitis who are treated with VPA or other HDAC inhibitors should be carefully monitored.

Infection of retinal neurons during murine cytomegalovirus retinitis.

PURPOSE: Previous results suggest that retinal neurons are infected early during murine cytomegalovirus (MCMV) infection of the inner retina. The purposes of this study were to identify which retinal neurons are infected and to determine the routes by which MCMV spreads in the retina. METHODS: Immunosuppressed (IS) BALB/c mice were inoculated with 5 x 10(3) PFU of MCMV (k181) through the supraciliary route. Injected eyes were collected at several times after inoculation, sectioned, and examined by electron microscopy and by staining for retinal cell antigens and for MCMV early (EA) or late (LA) antigen. RESULTS: MCMV-infected cells were observed in the choroid and RPE by day 3 after infection (PI) and in the inner retina beginning at day 5 PI. At this time, many horizontal and bipolar cells were MCMV-antigen-positive but only rare MCMV-infected amacrine cells (glycine positive or gamma-aminobutyric acid [GABA] positive) or MCMV-infected ganglion cells (NF positive) were observed in the inner retina. At day 10 PI, most virus-infected cells were glial fibrillary acidic protein (GFAP)- and GABA-positive glia. Virions were observed by electron microscopy in the choroid, RPE, and inner nuclear layer of the retina. Although virions were observed in the endothelium of the retinal vessels and the nearby retinal cells, the endothelial cell lining of the retinal vessels remained intact. Both apoptotic cells and necrotic cells were seen in the inner retina. CONCLUSIONS: In the inner retina, horizontal and bipolar cells were the early (< or = day 7 PI) targets of MCMV infection. Virus spread from the RPE and the photoreceptor layer to the inner retina through infected Muller cells and within the inner retina horizontally through infected horizontal cells.

Retroviral gene transfer into retinal pigment epithelial cells followed by transplantation into rat retina.

In this preliminary report, we describe a technique for gene transfer into the retina using a retrovirus vector. We transferred the bacterial LacZ gene and the neomycin-resistance gene into pigmented wild-type rat retinal pigment epithelial (RPE) cells in culture. The RPE culture was exposed to retrovirus, and infected cells were selected with a neomycin analog (G418). The LacZ gene product was detected by X-Gal histochemistry in 95-100% of drug-resistant cells. These genetically labeled cells were transplanted into the subretinal space of two 15- to 25-day-old albino RCS rats, which have an inherited retinal degeneration syndrome. The retinas were fixed and stained with X-Gal at 3 and 6 weeks after transplantation. At both time points, pigmented, LacZ-containing cells were seen in the subretinal space. Further, there were several rows of photoreceptor nuclei in the transplant area of the approximately 2-month-old rats, while in the control contralateral eye the photoreceptor nuclei were virtually absent, as for untreated animals, suggesting that the transplanted LacZ-marked, wild-type RPE cells may have helped preserve photoreceptors. The technique for gene transfer into RPEs followed by transplantation thus provides a means for gene therapy in organisms with a genetic defect in RPE cells.

Centripetal transport of herpes simplex virus in human retinal pigment epithelial cells in vitro.

Herpes simplex virus displays tropism for neurons and other polarized epithelial cells. We have grown human retinal pigment epithelial cells in culture to study potential mechanisms whereby herpes simplex virus (type I) is transported from the plasma membrane of the cell to the nucleus. The cells were highly polarized as determined by a variety of criteria. They were tightly coupled by junctional complexes, as determined by electron microscopy, immunofluorescent staining of tight junctions and measurements of transepithelial electrical resistances > 200 omega cm2. Immunofluorescence and confocal microscopy were used to visualize microtubule orientation. The microtubules were arranged (i) in a single apical cilium, (ii) in a meshwork beneath the apical membrane and (iii) in longitudinally arranged bundles near the lateral membranes and nucleus. The latter microtubules were primarily oriented with their plus ends directed toward the basal surface of the cells. We infected retinal pigment epithelial cells at the apical surface with virus and assayed the uptake and transport of virus to the nucleus by quantitative immunoblot and immunocytochemical staining for the viral immediate early gene product, infected cell protein 4. The antigen first appeared in retinal pigment epithelial cells 2 h after infection. Treatment of the cells with 33 microM nocodazole, a microtubule-destabilizing drug, delayed the appearance of the viral antigen by 1 h. The effect of nocodazole treatment on microtubule integrity was confirmed by immunofluorescent staining and immunoblots of tubulin. Both cytoplasmic dynein and the ubiquitous form of kinesin were identified in the cells using immunoblots. These novel data indicate that human retinal pigment epithelial cells, like neurons, are susceptible to infection by herpes simplex virus and that the centripetal transport of virus to the nucleus in both cell types is facilitated by microtubules. The orientation of microtubules in retinal pigment epithelial cells suggests that the transport of herpes simplex virus from the apical surface is mediated by a microtubule-activated motor enzyme, possibly kinesin.

Impaired killing of HCMV-infected retinal pigment epithelial cells by anti-pp65 CD8(+) cytotoxic T cells.

PURPOSE: Host defense against infection by human cytomegalovirus (HCMV) is ensured in great part by cytotoxic CD8(+) T lymphocytes (CTLs) directed against the tegument protein pp65. The hyperimmediate release of incoming pp65 into the major histocompatibility complex (MHC) class I pathway after fusion of the virus with the cell membrane provides a very early mechanism of defense. In retinal pigment epithelial (RPE) cells HCMV is known to enter through endocytosis. This study was conducted to determine whether this means of penetration into the cells would allow the virus to elude immune surveillance. METHODS: Infection of RPE cells with HCMV AD169 was performed for 6 hours, 48 hours, and 8 days. Expression of intracellular pp65 in RPE cells and in the astrocytoma reference cell line U373MG was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. Killing of both HCMV-infected cell lines by HLA-A2-restricted CD8(+) CTLs directed against pp65 was monitored by (51)Cr-release assays. RESULTS: RPE cells were not lysed by CTLs directed against incoming pp65, contrary to U373MG. Moreover, both cell lines were not killed by anti-pp65 CTLs later after infection, because of the MHC class-I-downregulating effect of HCMV unique short (US2-11) proteins. CONCLUSIONS: In RPE cells, both HCMV entry through endocytosis and the immunosuppressive effect of US proteins could allow the virus to evade immune surveillance at any stage of infection, which could promote viral spreading within the retina.

Cytomegalovirus replication in human retinal pigment epithelial cells. Altered expression of viral early proteins.

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 (10(6.5) TCID50/0.1 ml) significantly surpassed levels produced by HEL cells (10(5.5)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 in fibroblasts. 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.

Intraocular viral replication after systemic murine cytomegalovirus infection requires immunosuppression.

PURPOSE: Human cytomegalovirus retinitis is the most common blinding complication of acquired immune deficiency syndrome. However, the pathogenesis of the disease is poorly understood. The authors sought to characterize intraocular viral replication after systemic murine cytomegalovirus (MCMV) infection in the normal and immunosuppressed Balb/c mouse. METHODS: Normal or immunosuppressed mice (400 rads radiation plus antilymphocyte serum) were infected intravenously with a recombinant MCMV (RM408) that carries an MCMV IE1 promoter--LacZ insert. In vivo MCMV replication and its tissue distribution were monitored by beta-gal activity with x-gal staining on frozen tissue sections of multiple organs harvested from infected mice at different time points after inoculation. RESULTS: MCMV replication within the eye can be detected in the immunosuppressed Balb/c mouse but not in the normal host. Intraocular viral replication was noted first, and most frequently, in the ciliary body and was mainly restricted to the uveal tract. Intraocular viral replication coincided with the peak of systemic viral replication; however, the neurosensory retina was spared. In contrast, supraciliary inoculation of MCMV in the immunosuppressed Balb/c mouse resulted in massive viral replication and destruction of the neurosensory retina. CONCLUSIONS: This study demonstrated that intraocular MCMV replication after systemic infection requires systemic immunosuppression. Furthermore, the ciliary body is the portal of entry for the virus within the eye. MCMV can replicate in the epithelium of the uvea and retinal pigment epithelium, but it does not replicate within the neurosensory retina. The absence of MCMV replication within the neurosensory retina is not caused by either a defect in the recombinant virus or the inability of the host tissue to support viral replication.

Adenovirus-mediated gene transfer of ornithine aminotransferase in cultured human retinal pigment epithelium.

PURPOSE: To evaluate the efficacy of adenovirus mediated transfer of ornithine delta-aminotransferase (OAT) into human retinal pigment epithelial (RPE) cells. METHODS: Adenovirus-mediated gene transfer into primary cultures of human RPE was evaluated by measurement of enzyme activity in whole cell extracts and by Western blot analysis. To assess mitochondrial integrity, succinate dehydrogenase activity was measured in transduced RPE cells. Expression of adenovirus early genes was evaluated using reverse transcription-polymerase chain reaction. RESULTS: OAT activity, which was 65 nmol/mg.hour in untransduced cells, could be increased to levels in excess of 20,000 nmol/mg.hour using an adenovirus vector carrying the OAT cDNA. There was, however, a significant reduction in succinate dehydrogenase activity associated with OAT activity greater than 12,000 nmol/mg.hour. Transduced human RPE displayed an altered morphology that appears to be a response to the vector because similar changes could be induced by an adenovirus vector that does not carry the OAT cDNA. Adenovirus early gene expression was detected in transduced RPE. CONCLUSIONS: This study represents a first step in the development of intraocular gene replacement therapy for the treatment of gyrate atrophy. The authors demonstrate that adenovirus is an efficient vehicle for the delivery of OAT into human RPE and that RPE will tolerate greater than a 150-fold increase in OAT-specific activity. Evidence for disruption of mitochondria when OAT activity exceeds 12,000 nmol/mg.hour and vector-induced toxicity indicate that more controlled transgene expression and refinement of the vector systems is needed.