Redistribution and Reduction of Interphotoreceptor Retinoid-Binding Protein During Ocular Coronavirus Infection

Suson G. Robbins,* Barbara Wiggert.f Geetha Kutty,f Gerald J. Chader.f Barbara Derrick,* and John J. Hooks'1

Inoculation of the neurotropic coronavirus mouse hepatitis virus strain JHM intravitreally or into the anterior chamber causes acute infection of the retinal pigment epithelium (RPE) and neural retina. Weeks later, many retinas have foci of moderate to severe atrophy. The effect of coronavirus infection (after intravitreal inoculation) was examined on interphotoreceptor retinoid-binding protein (IRBP), the glycolipoprotein in the interphotoreceptor matrix (IPM) thought to transport retinoids between the photoreceptors and the RPE. Changes in IRBP distribution accompanied virus-associated retinal pathology, including photoreceptor loss and RPE abnormalities. Immunohistochemistry on days 3 and 6 showed that IRBP had diffused into the neural retina away from the IPM. The IRBP became localized abnormally in the same areas as virus-induced lesions, shown by staining adjacent sections with a monoclonal antibody specific for the viral nucleocapsid protein. Moreover, the level of IRBP in isolated retinas, measured in an immunoslot-blot assay, decreased significantly by day 3 and remained low through day 23. This decrease was confirmed in eyecups isolated on day 6. It may be caused in part by loss of photoreceptors and diffusion of IRBP through the retina into the vitreous. These studies show that a virus may induce an acute, limited infection in the retina that can be cleared by the host. However, the infection initiated a series of events resulting in long-term reduction and redistribution of a critical photoreceptor protein. Invest Ophthalmol Vis Sci 33:60-67,1992

The causes of many retinal degenerations are un- agents.1112 Small animal models of retinopathies in- known. For example, although usually considered a duced by such agents were developed for scrapie13 and group of "inherited" diseases, many forms of retinitis Creutzfeldt-Jakob disease.14"16 pigmentosa (RP) are sporadic, with no definable ge- Recently, we described a murine model of retinal netic component.1 A hallmark of some cases is the atrophy induced by intravitreal inoculation of the absence of detectable interphotoreceptor retinoid- neurotropic coronavirus, mouse hepatitis virus, strain binding protein (IRBP).2-5 JHM.910 This virus infects, not only the iris and cili- Viral infections were linked strongly to a small num- ary body, but also the retinal pigment epithelium ber of cases of rapid retinal degeneration in adults.6"10 (RPE) and neural retina. Focal degenerative changes It is conceivable that, in other cases, an earlier viral were observed by 3-4 weeks, even though the virus infection might have caused retinal cell defects whose was not detectable after 10 days. This model illus- consequences only appeared later. The slow time trates the potential of an acute viral infection to cause course and often focal nature of some of the RPs are later degenerative changes in the retina. consistent with this supposition. Moreover, a few In this study, we examined in greater detail the se- slowly progressive diseases of the central nervous sys- quelae of intravitreal injection of JHM virus into tem, previously thought to have a genetic basis, are mouse eyes. Along with morphologic changes, we now known to be caused by slow infectious charted the localization and amount of IRBP, because this protein can be used to assess, not only photoreceptor cell metabolic function, but also the integrity From the "Immunology and Virology Section, Laboratory of Im- 17 munology, and the f Laboratory of Retinal Cell and Molecular Biol- of the interphotoreceptor space or matrix (IPM). We ogy, National Eye Institute, National Institutes of Health, Bethesda, were interested especially in correlating changes in Maryland. IRBP and retinal pathology with detectability of the Submitted for publication: January 31, 1991; accepted July 25, viral nucleocapsid antigen. This would allow us to 1991. Reprint requests: John J. Hooks, PhD, National Eye Institute, establish the principle that a viral infection can under- National Institutes of Health, Building 10, Room 6N228, Bethesda, lie biochemical changes and, therefore, functional MD 20892. changes in the retina.

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Materials and Methods ice-cold PBS under the dissecting microscope. The cornea was removed by nicking with a razor blade Injections and Harvests then cutting with iris scissors. After extraction of the Adult (10-12-week old) male BALB/c mice were lens, the neural retinas were removed and placed in injected intravitreally with 1.5 X 104 plaque-forming Nunc cryotubes (Nunc Intermed, Denmark) on ice. units of mouse hepatitis virus (strain JHM) or with The retinas were stored at -70°C for later quantita- supernatant from mock-inoculated cell cultures. The tionoflRBP. eyes processed as described previously9 after harvest- ing on days 3, 6, and 21 or 23 after inoculation. The Preparation of Eyecups mice were anesthetized as described9 and killed by Both eyes of two uninjected and six to nine injected decapitation (Harvard Apparatus, South Natick, mice were used for eyecups. Each eye was rinsed, blot- MA). They were handled according to the ARVO Res- ted, and placed in its natural orientation on a plastic olution on the Use of Animals in Research. The virus- tissue culture dish on a flat bed of dry ice. It was fro- injected (left) eye was removed and dissected first, as zen for 5 min, allowing the eye to adhere to the dish. quickly as possible. The mock-injected (right) eye was The cornea was removed by slicing vertically with a placed in situ against a plastic sheet on ice until it razor blade. The dish was placed under the dissecting could be processed. On each harvest day, the eyes of microscope under minimum illumination, and the two mice were fixed in glutaraldehyde, and two were eye was thawed partially, lens side up, until the lens frozen in O.C.T. (Miles, Inc., Elkhart, IN). could be removed with forceps. Each resulting eyecup was frozen in a Nunc cryotube on dry ice and stored at Antisera -70°C for later quantitation of IRBP. For detection of IRBP, an affinity-purified polyclo- Quantitation of IRBP by Slot-Blot Analysis nal antiserum prepared in goats against bovine IRBP1819 was used on acetone-fixed frozen sections at Individual retinas or eyecups were homogenized in a 1:100 dilution;20 affinity-purified normal goat im- 10 mM Tris buffer, pH 7.5, containing 0.5 M NaCl munoglobulin G was used as a control (Organon, and 2 mM ethylenediaminetetraacetic acid. The solu- West Chester, PA). A murine monoclonal antibody ble fraction was prepared by centrifugation at 110,000 (H3B5, in the form of purified ascites fluid) raised g for 60 min. Slot-blot analysis was done as previously described.20 Protein concentrations were deter- against human IRBP was provided generously by Dr. 22 Larry Donoso (Wills Eye Hospital, Philadelphia, PA). mined by the Bradford dye-binding method using a It was used on acetone-fixed slides at a 1:100 dilution. micro Bio-Rad (Richmond, CA) protocol. Our results An unrelated murine monoclonal ascites antibody are expressed in nanograms or micrograms of IRBP (PR8) with specificity for rat photoreceptors (devel- (bovine IRBP equivalents) per milligram of total su- oped and characterized by J.J. Hooks and B. Detrick; pernatant protein. NIH, Bethesda, MD) was used as a control. Results Redistribution of IRBP in Infected Eyes Immunocytochemistry The virus-associated effects on the retina after in- Adjacent frozen sections were assayed immunologi- travitreal inoculation of 10-week-old BALB/c mice cally for IRBP and the viral nucleocapsid antigen us- 21 with JHM virus are depicted in Figure 1. A hematoxy- ing an immunoperoxidase-based assay. The pri- lin and eosin stain of a normal retina from an unin- mary antibodies were those listed. As secondary anti- jected mouse is shown in Figure 1 A. To localize IRBP bodies after incubation with anti-IRBP, we used in normal eyes, frozen sections were stained with poly- biotinylated rabbit anti-goat immunoglobulin G for clonal antibody specific for bovine IRBP in immuno- detecting goat anti-bovine IRBP and horse anti- peroxidase assays. The staining pattern of IRBP in a mouse (rat-adsorbed) immunoglobulin G for detect- normal eye (mock injected, day 3) is shown in Figure ing IRBP-specific monoclonal antibodies. 1C. Staining was highly specific; the only area in the Preparation of Retinas retina which reacted with the antibody was the IPM. The same normal pattern was observed 1 day after Neural retinas were obtained as follows. Both eyes inoculation. No staining was present after reacting of two to three uninjected mice and six to seven in- sections of normal eyes with goat immunoglobulin G jected mice were harvested. Each eye was rinsed with (Fig. IB). phosphate-buffered saline (PBS) and placed in a plas- By day 3, significant pathologic changes were evi- tic tissue culture dish (3-cm diameter) with 3 ml of dent (Fig. ID). The retinas of the two eyes examined

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Fig. 1. Pathologic changes and redistribution of IRBP in retinas infected with JHM virus. Eyes were harvested on days 3 and 6, and fixed and sectioned for hematoxylin and eosin (H&E) staining, or frozen and sectioned for immunostaining with IRBP-specific (or control) antibody. Panels (a-c), sections of mock-injected eyes, stained with H&E (a), goat IgG (b), and polyclonal anti-IRBP antiserum (c). (d-f) Virus-injected eyes, day 3, stained with H&E (d), goat IgG (e), and polyclonal anti-IRBP antiserum (f). (g-i) Virus-injected eyes, day 6, stained with H&E (g), goat IgG (h), and anti-IRBP (i). Original magnification X50.

showed five focal lesions restricted to the posterior had diffused in the direction of the vitreous, reaching (observed at the plane of the optic nerve). These as far as the inner nuclear layer. In the retina shown, usually involved the ganglion cell and inner nuclear there were approximately five lesions (one a cluster of layers and sometimes the photoreceptors and RPE. Muller-like fibers, shown in the figure). These lesions The nuclear layers often were disorganized from a loss were scattered throughout the retina, with one located of nuclei, and outer segments were missing. Focal at the periphery. Their total area was approximately hemorrhage occurred, affecting both nuclear layers 15% (observed at a plane near the optic disc). In the and the interphotoreceptor space (one of two eyes). second retina, approximately one half (one "side" in There was moderate inflammation of the posterior section) showed abnormal staining for IRBP. Areas pole, with polymorphonuclear neutrophils and mac- where IRBP had diffused usually corresponded to rophages infiltrating the ganglion cell and inner plexi- areas with virus-associated disruption. In particular, form layers (two of two eyes). Approximately one half the stain often followed the path of fibers that proba- of the nuclei in the regions of the ganglion cell in the bly represented Muller cell bodies. The IRBP-specific lesions were pyknotic. The RPE appeared vacuolated, stain also accumulated immediately above the RPE unlike the RPE in uninjected or mock-injected eyes. layer and in large clumps from the IPM to the inner Striking changes in IRBP staining were seen in fro- nuclear layer. Less stain was present in the outer seg- zen sections from eyes injected with JHM virus and ment area than in the controls. These patterns proba- harvested on day 3 postinjection (Fig. IF). The IRBP bly represented leakage of IRBP past a disrupted outer was not restricted to the photoreceptor region around limiting membrane, into necrotic areas, and diffusion the outer segments and subretinal space but, instead, into infected and damaged cells. Similar results were

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No. 1 VIRUS-INDUCED IRBP ABNORMALITY / Robbins er ol

obtained using a monoclonal antibody specific for sections had diminished greatly from that seen on day IRBP. 3, corroborating the results of quantitative assays. No On day 6 after inoculation of virus, there was in- background staining was present in virus-injected creased disruption of the retina, with all layers af- eyes reacted with nonimmune goat immunoglobulin fected and major areas of detachment (Fig. 1G). In the G (Fig. 1H). These results were confirmed using the eye shown, there were severe changes in approxi- monoclonal antibody specific for IRBP. mately 40% of the retina and moderate changes in approximately 10%. In the second eye, approximately Correlation of IRBP Alterations and Viral Antigen 95% of the retina was affected profoundly, with only 5% mildly abnormal. In both eyes, the photoreceptor Viral nucleocapsid antigen was detected in frozen and RPE cells were affected severely. Moderate num- sections of virus-injected eyes, using a monoclonal bers of macrophages had infiltrated the nuclear layers antibody specific for the JHM viral nucleocapsid pro- (not shown). There were no detectable pathologic tein, as reported previously.910 The results showed changes in the anterior segment. that, by day 3, mainly Muller-like cells and ganglion When sections of a virus-injected eye from day 6 cells were infected (Fig. 2C). Small areas of the RPE were reacted with IRBP-specific antibody, the result also stained. In both eyes harvested on day 3, approxi- was a diffuse light stain throughout the neural retina, mately 20% of photoreceptor cells appeared to be in- including the IPM (Fig. 11). In the eye shown, approxi- fected, lying in strands along infected Muller-like mately 75% of the IRBP was displaced from the IPM fibers. In the eye shown, there were approximately 20 (approximately one half of the posterior was unaf- of these lesions, scattered over approximately 60% of fected). The total amount of IRBP visible in these the area of the retina. In the other eye, there were

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Fig. 2. Distribution of viral nucleocapsid antigen in JHM virus-infected retinas, detected by immunoperoxidase assay, (a) Retina of virus-injected eye, day 3, incubated with normal mouse IgG. (b) Retina of mock-injected eye, day 6, incubated with monoclonal anti-viral nucleocapsid antibody, (c) Retina of virus-injected eye, day 3, incubated with monoclonal anti-viral nucleocapsid antibody. Staining in this field is restricted to Muller-like cells (some RPE cells in other areas were also positive), (d) Retina of virus-injected eye, incubated with monoclonal anti-viral nucleocapsid antibody. Both inner and outer nuclear layers were positive. Original magnification X50.

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about 12 such foci, making up approximately 40% of results. On day 3 (Fig. 4), IRBP decreased from an the retina (mainly one "side"). average of 496 ng/mg protein in retinas from mock- By day 6, about 12 foci of infection continued to be injected eyes to 283 ng/mg in retinas from virus-in- scattered over approximately 75% of the retina. The jected eyes. This decrease in IRBP continued through pattern was partly Muller-like and partly restricted to day 6 (522 versus 319 ng/mg) and day 23 (678 versus the photoreceptor, inner nuclear, or ganglion cell 311 ng/mg). This change was highly significant by the layers (Fig. 2D). Photoreceptor "ghosts" of nuclei student t-test (P < 0.001, combined data from all were present in one lesion, surrounded by infected three times). The injection process alone caused an Muller fibers. No viral antigen was detectable on initial drop of over 50% (1076 versus 496 ng/mg pro- day 21. tein in uninjected versus mock-injected retinas). This By staining adjacent sections (day 6), we showed difference lessened gradually over 3 weeks, at which that most areas where IRBP had diffused into the ret- time the IRBP values for uninjected and mock-in- ina (Fig. 3B) corresponded closely with areas positive jected retinas overlapped. for viral nucleocapsid antigen (Fig. 3D). Incubation of The results from eyecups harvested on day 6 were sections from a virus-injected eye with normal goat as significant as for retinas (P < 0.001, Fig. 5). The (Fig. 3A) or mouse immunoglobulin G (Fig. 3C) IRBP fell from 12.87 (mock-injected) to 3.53 (virus- yielded no background in the retina. Neither did incu- injected) jig/mg protein. However, on other days (day bation of sections from a mock-injected eye after in- 1, 3, and 21), the differences between virus-injected cubation with normal mouse sera (not shown). and uninjected or mock-injected eyes were not significant. This was probably caused by the large ratio of Quantitation of IRBP in Retinas and Eyecups total protein to IRBP. The results from retinas and eyecups taken together showed that the virus-asso- The IRBP in retinas and eyecups was determined ciated effect on IRBP levels on day 6 was reflected, by quantitative immunoblot assay, with the following not only in the IRBP associated with photoreceptors,

Fig. 3. Correlation of IRBP redistribution with virus-induced lesions. Frozen sections of virus-injected eyes harvested on day 6 p.i. were incubated with goat IgG (a), polyclonal anti-IRBP

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IRBP in Retinas £ 1200

Fig. 4. Reduction of IRBP concentration in retinas after intravitreal injection of JHM 1000- virus. Retinas were harvested from uninjected, mock-injected, and virus-injected 800- Uninjected mice on days 3, 6, and 23. Corneas were ex- Mock-injected cised, the lenses removed, and the retinas Virus-injected tested for IRBP in a quantitative immuno- 600- slot-blot assay. IRBP concentration is expressed as nanograms/milligram protein. Ver- 400- tical bars indicate standard errors of the mean. 200

but also in the entire pool of IRBP, ie, that harvested The effect of viral infection on IRBP was not previ- with the isolated retina and that located in the IPM. ously evaluated to our knowledge. A large lipoglyco- protein, IRBP is synthesized by retinal photoreceptor 2324 Discussion cells and secreted into the IPM20-23-25"29 where it constitutes a major percentage of the readily soluble We established an animal model of virus-induced 910 proteins. It serves the important function of transport- retinal disease. Murine coronavirus can replicate ing retinoids between the photoreceptors and RPE in the retina, produce an acute necrotizing disease of cells.17 We found that levels of IRBP were decreased the sensory retina, and induce long-term sequelae. A markedly in virus-infected retinas in comparison with potentially important characteristic of the disease is normal or mock-infected retinas. This difference was the persistence of retinal damage in the absence of apparent 3 days after infection and persisted through detectable virus. a time (day 23) when virus was no longer present. The mechanism by which the virus decreased the IRBP Retinas Eyecups level is unknown. One possibility is that the virus in- terfered with normal synthesis and/or secretion of IRBP through the rough endoplasmic reticulum and 1200 - 25 Golgi complex of the photoreceptor cell. However, c 1000 although these virions are known to use a cell's nor- "i i- 20 mal secretory pathway as an exit route, there is no 800 evidence that viral infection blocks macromolecular 15 synthesis or secretion of proteins in other cell 22 Proteir loncen 3031 O O_ 600 types. Diminished IRBP concentration seemed to 10 correlate with early retinal infection (day 3), with dis- 5 400

IRI organization of the neural retina, and with abnormali- c — (ug/ 5 ties in the RPE. This temporal relationship and the a> 200 viean finding that IRBP was somewhat reduced in mock-in- 0 0 fected retinas, but returned to normal levels after 3 weeks, indicated that the IRBP concentration was perturbed by insults, such as viral infection and intra- • Uninjected 0 Mock-Injected ocular injection, and that IRBP levels are probably a M Virus-Injected sensitive general indicator of photoreceptor cell health. Experiments are planned to assess other im- Fig. 5. Reduction of IRBP in eyecups compared to retinas, har- portant biochemical components in the retina that vested 6 days after inoculation of JHM virus. Retinas were obtained as described in Figure 4. Eyecups were prepared by freezing each might be affected in this disease process and the early eye on dry ice, slicing off the cornea, and removing the lens. IRBP effects on electrophysiologic parameters. levels were determined by immuno-slot-blot analysis. Note that IRBP values for retinas are expressed as nanograms/milligram pro- Along with the quantitative decrease, we found that tein, whereas values for eyecups are in micrograms/milligram pro- IRBP distribution was associated with abnormal im- tein. munostaining of IRBP after the localization of virus-

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induced disruption of the adhering junctions between Chader GJ: Dominantly inherited retinitis pigmentosa: Ultra- photoreceptor and Muller glial cells. This may indi- structure and biochemical analysis. Ophthalmology 92:1165, 1985. cate an important abnormality in the localization and 6. Culbertson WW, Blumenkranz MS, Pepose JS, Stewart JA, distribution of a number of IPM components that and Curtin VT: Varicella zoster virus is a cause of the acute could lead to secondary pathologic effects. For exam- retinal necrosis syndrome. Ophthalmology 93:559, 1986. ple, with IRBP both diminished in concentration and 7. Deutman AF and Klomp HJ: Rift Valley fever retinitis. Am J no longer in its usual position between RPE and pho- Ophthalmol 92:38, 1981. toreceptor cells, retinoid transport might be disrupted. 8. de Venezia G, Zu Rhein GM, Pratt MV, Kisken W: Cytomega- lic inclusion retinitis in an adult. Arch Ophthalmol 86:44, The resultant retinoid excess in the photoreceptor 1971. could be toxic to the cell. Future work is necessary to 9. Robbins SG, Hamel CP, Detrick B, and Hooks JJ: Murine determine if other important IPM components simi- coronavirus induces an acute and long-lasting disease of the larly diffuse out of their usual IPM locale. retina. Lab Invest 62:417, 1990. 10. Robbins, SG, Detrick, B and Hooks, JJ: Ocular tropisms of One of the most intriguing aspects was that no viral murine coronavirus (strain JHM) after inoculation by various antigen could be detected in affected retinas 3 weeks routes. Invest Ophthalmol Vis Sci 32:1883, 1991. 910 after infection. Thus, viral "footprints" disappear 11. Gajdusek DC: Unconventional viruses and the origin and dis- at an active stage in the disease, before the degenera- appearance of kuru. Science 197:943, 1977. tive process is complete. It is not known if virus-in- 12. 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