Investigative Ophthalmology & Visual Science, Vol. 33, No. 1, January 1992 Copyright © Association for Research in Vision and Ophthalmology 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 pa- thology, 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 photore- ceptor 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. 60 Downloaded from iovs.arvojournals.org on 09/28/2021 No. 1 VIRUS-INDUCED IRDP ABNORMALITY / Robbins er ol 61 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 previ- ously 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 Downloaded from iovs.arvojournals.org on 09/28/2021 62 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1992 Vol. 33 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).