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.

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    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.
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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.

Item Type: Thesis (Doctoral (PhD))
URI: http://research.library.mun.ca/id/eprint/5684
Item ID: 5684
Additional Information: Bibliography: leaves [226]-253.
Department(s): Medicine, Faculty of
Date: 1992
Date Type: Submission
Library of Congress Subject Heading: Diabetes--Pathogenesis; Viruses--Receptors; Pancreatic beta cells
Medical Subject Heading: Diabetes Mellitus, Type 1; Receptors, Virus; Islets of Langerhans; Encephalomyocarditis virus

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