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Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan.

Kunz S, Sevilla N, McGavern DB, Campbell KP, Oldstone MB - J. Cell Biol. (2001)

Bottom Line: In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection.We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV.This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Division of Virology, Department of Neuropharmacology, La Jolla, CA 92037, USA.

ABSTRACT
alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.

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Biochemical characterization of the LCMV–α-DG interaction. α-DG purified from rabbit skeletal muscle or enriched from MC57 cells was subjected to VOPBA using 107 PFU/ml of LCMV clone-13 or to laminin overlay assay with 10 μg/ml biotinylated laminin-1. Bound virus was detected using monoclonal antibodies WE33 and WE36 against LCMV-GP. For detection of bound biotinylated laminin-1, HRP- conjugated streptavidin was used. (A) The cation dependence of the binding of LCMV and laminin-1 to α-DG was tested by addition of 1 mM Ca2+, 1 mM Mg2+, 10 mM EDTA, or 10 mM EGTA. (B) For inhibition with heparin, virus or biotinylated laminin-1 were pretreated with 0, 25, and 100 mg/ml heparin before incubation with immobilized α-DG. (C) To test for a potential role of α-DG–derived carbohydrates in virus binding, α-DG from MC57 cells was pretreated with 0, 1, 10, and 100 mM sodium periodate before the addition of virus. A potential role of α-DG–derived terminal sialic acids was addressed by neuraminidase treatment (D) and competition with 0, 10, and 100 mM sialic acid (E).
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fig1: Biochemical characterization of the LCMV–α-DG interaction. α-DG purified from rabbit skeletal muscle or enriched from MC57 cells was subjected to VOPBA using 107 PFU/ml of LCMV clone-13 or to laminin overlay assay with 10 μg/ml biotinylated laminin-1. Bound virus was detected using monoclonal antibodies WE33 and WE36 against LCMV-GP. For detection of bound biotinylated laminin-1, HRP- conjugated streptavidin was used. (A) The cation dependence of the binding of LCMV and laminin-1 to α-DG was tested by addition of 1 mM Ca2+, 1 mM Mg2+, 10 mM EDTA, or 10 mM EGTA. (B) For inhibition with heparin, virus or biotinylated laminin-1 were pretreated with 0, 25, and 100 mg/ml heparin before incubation with immobilized α-DG. (C) To test for a potential role of α-DG–derived carbohydrates in virus binding, α-DG from MC57 cells was pretreated with 0, 1, 10, and 100 mM sodium periodate before the addition of virus. A potential role of α-DG–derived terminal sialic acids was addressed by neuraminidase treatment (D) and competition with 0, 10, and 100 mM sialic acid (E).

Mentions: Since previous studies suggest a lectin-type binding for all α-DG–ECM protein interactions described so far, we evaluated to what extent the LCMV–α-DG interaction shares these biochemical characteristics. To address this question, virus overlay protein binding assays (VOPBAs) were performed in the presence and absence of divalent cations, chelating agents, and heparin using α-DG purified from skeletal muscle and α-DG–enriched glycoprotein fractions isolated from MC57 cells. For comparison, a laminin-1 overlay binding assay was performed. In contrast to α-DG–laminin-1 binding and other α-DG ECM protein interactions, the binding of LCMV to α-DG was independent of divalent cations and insensitive to the chelating agents EDTA and EGTA (Fig. 1 A). Further, the presence of ≥100 μg/ml heparin blocked laminin-1 binding completely but did not significantly affect virus binding (Fig. 1 B). Evidently, this virus–α-DG binding was biochemically distinct from the interactions between α-DG and ECM proteins. Similarly, α-DG–derived sialic acids were not involved in virus binding despite their apparent role in α-DG–laminin binding (Yamada et al., 1996). Periodate treatment of α-DG, under conditions in which primarily terminal sialic acid residues but not core glycan structures are oxidized, had no effect on virus binding (Fig. 1 C). However, with more extensive deglycosylation a reduction in virus binding was achieved, consistent with previous findings (Cao et al., 1998). Further, treatment of α-DG with neuraminidase (Fig. 1 D) and competition with soluble sialic acid, treatments that markedly reduced laminin–α-DG binding in some studies (Yamada et al., 1996), had no effect on the virus–α-DG interaction. These findings indicate that the binding of LCMV to α-DG differs notably from the binding of α-DG to laminin, agrin, or perlecan.


Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan.

Kunz S, Sevilla N, McGavern DB, Campbell KP, Oldstone MB - J. Cell Biol. (2001)

Biochemical characterization of the LCMV–α-DG interaction. α-DG purified from rabbit skeletal muscle or enriched from MC57 cells was subjected to VOPBA using 107 PFU/ml of LCMV clone-13 or to laminin overlay assay with 10 μg/ml biotinylated laminin-1. Bound virus was detected using monoclonal antibodies WE33 and WE36 against LCMV-GP. For detection of bound biotinylated laminin-1, HRP- conjugated streptavidin was used. (A) The cation dependence of the binding of LCMV and laminin-1 to α-DG was tested by addition of 1 mM Ca2+, 1 mM Mg2+, 10 mM EDTA, or 10 mM EGTA. (B) For inhibition with heparin, virus or biotinylated laminin-1 were pretreated with 0, 25, and 100 mg/ml heparin before incubation with immobilized α-DG. (C) To test for a potential role of α-DG–derived carbohydrates in virus binding, α-DG from MC57 cells was pretreated with 0, 1, 10, and 100 mM sodium periodate before the addition of virus. A potential role of α-DG–derived terminal sialic acids was addressed by neuraminidase treatment (D) and competition with 0, 10, and 100 mM sialic acid (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198839&req=5

fig1: Biochemical characterization of the LCMV–α-DG interaction. α-DG purified from rabbit skeletal muscle or enriched from MC57 cells was subjected to VOPBA using 107 PFU/ml of LCMV clone-13 or to laminin overlay assay with 10 μg/ml biotinylated laminin-1. Bound virus was detected using monoclonal antibodies WE33 and WE36 against LCMV-GP. For detection of bound biotinylated laminin-1, HRP- conjugated streptavidin was used. (A) The cation dependence of the binding of LCMV and laminin-1 to α-DG was tested by addition of 1 mM Ca2+, 1 mM Mg2+, 10 mM EDTA, or 10 mM EGTA. (B) For inhibition with heparin, virus or biotinylated laminin-1 were pretreated with 0, 25, and 100 mg/ml heparin before incubation with immobilized α-DG. (C) To test for a potential role of α-DG–derived carbohydrates in virus binding, α-DG from MC57 cells was pretreated with 0, 1, 10, and 100 mM sodium periodate before the addition of virus. A potential role of α-DG–derived terminal sialic acids was addressed by neuraminidase treatment (D) and competition with 0, 10, and 100 mM sialic acid (E).
Mentions: Since previous studies suggest a lectin-type binding for all α-DG–ECM protein interactions described so far, we evaluated to what extent the LCMV–α-DG interaction shares these biochemical characteristics. To address this question, virus overlay protein binding assays (VOPBAs) were performed in the presence and absence of divalent cations, chelating agents, and heparin using α-DG purified from skeletal muscle and α-DG–enriched glycoprotein fractions isolated from MC57 cells. For comparison, a laminin-1 overlay binding assay was performed. In contrast to α-DG–laminin-1 binding and other α-DG ECM protein interactions, the binding of LCMV to α-DG was independent of divalent cations and insensitive to the chelating agents EDTA and EGTA (Fig. 1 A). Further, the presence of ≥100 μg/ml heparin blocked laminin-1 binding completely but did not significantly affect virus binding (Fig. 1 B). Evidently, this virus–α-DG binding was biochemically distinct from the interactions between α-DG and ECM proteins. Similarly, α-DG–derived sialic acids were not involved in virus binding despite their apparent role in α-DG–laminin binding (Yamada et al., 1996). Periodate treatment of α-DG, under conditions in which primarily terminal sialic acid residues but not core glycan structures are oxidized, had no effect on virus binding (Fig. 1 C). However, with more extensive deglycosylation a reduction in virus binding was achieved, consistent with previous findings (Cao et al., 1998). Further, treatment of α-DG with neuraminidase (Fig. 1 D) and competition with soluble sialic acid, treatments that markedly reduced laminin–α-DG binding in some studies (Yamada et al., 1996), had no effect on the virus–α-DG interaction. These findings indicate that the binding of LCMV to α-DG differs notably from the binding of α-DG to laminin, agrin, or perlecan.

Bottom Line: In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection.We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV.This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Division of Virology, Department of Neuropharmacology, La Jolla, CA 92037, USA.

ABSTRACT
alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.

Show MeSH
Related in: MedlinePlus