Identification of α-fodrin as an autoantigen in experimental coronavirus retinopathy (ECOR)

Abstract

The coronavirus, mouse hepatitis virus (MHV), JHM strain induces a biphasic disease in BALB/c mice that consists of an acute retinitis followed by progression to a chronic retinal degeneration with autoimmune reactivity. Retinal degeneration resistant CD-1 mice do not develop either the late phase or autoimmune reactivity. A mouse RPE/choroid DNA expression library was screened using sera from virus infected BALB/c mice. Two clones were identified, villin-2 protein and α-fodrin protein. α-Fodrin protein was used for further analysis and western blot reactivity was seen only in sera from virus infected BALB/c mice. CD4 T cells were shown to specifically react with MHV antigens and with α-fodrin protein. These studies clearly identified both antibody and CD4 T cell reactivities to α-fodrin in sera from virus infected, retinal degenerative susceptible BALB/c mice.

Keywords: Autoantibodies; Autoimmunity; Coronavirus; Retinal degeneration; α-Fodrin.

Fig. 1

Immunoperoxidase staining of rat retina with non-pooled sera from uninjected, mock injected or MHV JHM injected BALB/c mice. Cryosections of normal rat retina were fixed and permeabilized with acetone/methanol and reacted with 1:40 dilutions of sera from individual (A) un-injected, (B) mock-injected BALB/c and (C–F) MHV JHM injected BALB/c mice. Sections were counter-stained with methyl green producing blue-green colored nuclei in the retina.

Fig. 2

Immunoperoxidase staining of rat retina with pooled sera from uninjected, mock injected or MHV JHM injected CD-1 mice. Cryosections of normal rat retina were fixed and permeabilized with acetone/methanol and reacted with 1:40 dilutions of pooled sera from (A) un-injected, (B) mock-injected CD-1 mice or (C) MHV JHM injected CD-1 mice. Sections were counter-stained with methyl green producing blue-green colored nuclei in the retina.

Fig. 3

Western blot analysis of murine, bovine and rat retinal proteins (A) or rat and bovine RPE proteins (B) with sera from control or MHV JHM infected BALB/c mice. Twenty micrograms of a retinal protein homogenate derived from either BALB/c mice or bovine eyes (A) or 20 μg of RPE protein homogenate derived from either Brown Norway rat or bovine eyes (B) was subjected to SDS-PAGE electrophoresis and then transferred to a nitrocellulose membrane. The protein blots were then reacted with pooled sera from control BALB/c mice or pooled sera from MHV JHM infected BALB/c mice with retinal degeneration. The arrows indicate proteins reactive with sera from mice infected with virus but not reactive to sera from control mice.

Fig. 4

Alignments of amino acid translation of the cDNA clones with α-fodrin and villin 2. (A) Alignment of the amino acid sequences for mouse α-fodrin and clone 11G (cl 11G). (B) Alignment of the amino acid sequences for mouse villin 2 and clone 8 (cl 8). Colons indicate identical amino acid residues and dots indicate conservative substitution of amino acid residues.

Fig. 4

Alignments of amino acid translation of the cDNA clones with α-fodrin and villin 2. (A) Alignment of the amino acid sequences for mouse α-fodrin and clone 11G (cl 11G). (B) Alignment of the amino acid sequences for mouse villin 2 and clone 8 (cl 8). Colons indicate identical amino acid residues and dots indicate conservative substitution of amino acid residues.

Fig. 5

Western blot analysis demonstrating immunoreactivity of sera from MHV JHM infected BALB/c mice with an α-fodrin peptide. Twenty-five nanograms of purified mouse α-fodrin peptide was loaded in each lane and subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. The protein blots were then reacted with sera from control (C), mock-injected (M) and virus-injected (V) BALB/c (A) and CD-1 (B) mice. Molecular weight standards are indicated on the left of the blots. The black arrow indicates reactivity of the α-fodrin peptide with sera from virus-injected BALB/c mice.

Fig. 6

(A) Effects of phytohemagglutinin (PHA; 1 μg/well), UV–inactivated mouse hepatitis virus (UV–MHV; 2 × 10⁵ PFU/well) and purified α-fodrin peptide (10 μg/well) on proliferation of splenocytes from MHV infected BALB/c mice (black bars) and uninfected BALB/c mice (gray bars). The bars represent the mean fold change in splenocyte proliferation ± SEM for each group. The data shown here is representative of two separate experiments, and three to six animals were in each group for each experiment. Treatment with PHA resulted in significant increases in splenocyte proliferation for both MHV infected (p = 0.02) and uninfected (p = 0.004) BALB/c mice. Incubation with UV–MHV significantly increased splenocyte proliferation only for MHV infected BALB/c mice (p < 0.0001). Splenocytes from MHV infected BALB/c mice (p = 0.04) responded to a peptide of α-fodrin, whereas, splenocytes from uninfected BALB/c mice did not respond. (B) Effects of a peptide of α-fodrin (10 μg/well) on proliferation of splenocyte populations enriched for CD4⁺ T cells, adherent cells or B cells from MHV infected BALB/c mice (black bars) and uninfected BALB/c mice (gray bars). Treatment with an α-fodrin peptide resulted in increases in proliferation of CD4⁺ T cells (p = 0.01) and adherent cells (p = 0.04) but not of B cells. Results were normalized by arbitrarily setting the change in proliferation of untreated cells to 1.0.