Characterization and isolation of a β-cell surface receptor for diabetogenic EMC-D virus and studies on the relationship between receptor number and pathogenesis in virus-induced IDDM in mice

Baldeh, Mansajang P. (1992) Characterization and isolation of a β-cell surface receptor for diabetogenic EMC-D virus and studies on the relationship between receptor number and pathogenesis in virus-induced IDDM in mice. Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (32MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

The diabetogenic variant of EMC virus, EMC-D, can infect mouse pancreatic β-cells and may induce a type 1 diabetes-like syndrome in SJL/J mice, but not in C57BL/6J mice. A number of reports suggest an association between pathogenicity and the number of β-cell receptors available for EMC-D virus binding. Yet a great deal more is known about EMC-D virus, than about the receptor structures that allow its entry into β-cells. The aim of this project was to characterize the virus receptor on BMP cells and to see if the receptor plays a role in EMC-D virus-induced diabetes in SJL/J mice. -- The nature of the EMC-D virus receptor was investigated by in vitro attachment of ³H-labelled EMC-D virus to BMP cells. Virus binding was saturable and reached equilibrium after 2 h at 4°C. Although Scatchard analysis revealed only a single high affinity (Kd = 1.2 nM; 4 X l0⁵ sites/cell) binding site, Hill analysis of the saturation binding data strongly suggested that EMC-D virus has more than one binding site on BMP cells. Pretreatment of the cells with protease, neuraminidases, or with lectins, showed that the receptor is a protein and that sialic acids are required for virus binding to BMP cells. Pretreating cells with phospholipases C and D or with enzymes specific for N-linked carbohydrates had no significant effect on virus binding. BMP cell surface receptor regeneration after protease treatment took about 6 h, and was inhibited by cycloheximide, but not by tunicamycin. HPLC analyses of total and neuraminidase-releasable sialic acids suggested that about 30% of the total sialic acid on BMP cells is Neu5Ac; Neu5Gc on BMP cells seemed resistant to V. cholerae neuraminidase. It is concluded that the receptor for EMC-D virus on BMP cells is a sialylated glycoprotein, and virus binding involves Neu5Ac, and possibly Neu5Gc, but not N-linked sugars. -- The Hill analysis result is supported by the detection of two affinity purified putative receptor proteins (M.W. 97,000 & 70,000 daltons) from BMP cells. Chromatofocusing studies on these proteins confirmed that the putative virus receptor contains Neu5Ac. Monoclonal antibodies raised against the putative receptor blocked EMC-D virus infection of BMP cells and virus binding to β-cells from SJL/J mice. FACS analyses of virus binding and anti-EMC virus anti-idiotypic antibody binding to β-cells, showed that cells from SJL/J and C57BL/6J mice contain about the same number of EMC-D virus receptors. It is concluded that the inability of EMC-D virus to cause IDDM in C57BL/6J may not be due to lack of, or a reduction in the number of virus receptors on β-cells from C57BL/6J mice. It is speculated that anti-idiotypic antibodies may play a role in the initiation of β-cell destruction in virus-infected SJL/J mice, possibly by molecular mimicry.

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