Posttranslational modification of alpha-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding

Abstract

The receptor for lymphocytic choriomeningitis virus (LCMV), the human pathogenic Lassa fever virus (LFV), and clade C New World arenaviruses is alpha-dystroglycan (alpha-DG), a cell surface receptor for proteins of the extracellular matrix (ECM). Specific posttranslational modification of alpha-DG by the glycosyltransferase LARGE is critical for its function as an ECM receptor. In the present study, we show that LARGE-dependent modification is also crucial for alpha-DG's function as a cellular receptor for arenaviruses. Virus binding involves the mucin-type domain of alpha-DG and depends on modification by LARGE. A crucial role of the LARGE-dependent glycosylation of alpha-DG for virus binding is found for several isolates of LCMV, LFV, and the arenaviruses Mobala and Oliveros. Since the posttranslational modification by LARGE is crucial for alpha-DG recognition by both arenaviruses and the host-derived ligand laminin, it also influences competition between virus and laminin for alpha-DG. Hence, LARGE-dependent glycosylation of alpha-DG has important implications for the virus-host cell interaction and the pathogenesis of LFV in humans.

FIG. 1.

The N-terminal region of the mucin-type domain of α-DG is necessary and sufficient for virus binding. (A) Furin recognition site of α-DG and schematic representation of the α-DG-Fc fusion proteins. The N-terminal domain (white), the mucin-type domain (black), and the C-terminal domain (gray) of α-DG, β-DG, and human IgG1Fc are indicated. SP, signal peptide. (B) Proteolytic processing of DGFc3 through -5. α-DG-Fc fusion proteins were purified from cell lysates or culture supernatant of transiently transfected HEK293T cells. Purified proteins were separated by SDS-PAGE under reducing conditions and analyzed by Western blot analysis using an anti-human IgG Fc antibody. Molecular masses are indicated to the left of the gels. (C) Virus binding to α-DG-Fc fusion proteins. Equal amounts of DGFc1 and -2 purified from cell lysates and DGFc3 through -5 purified from supernatants were separated by SDS-PAGE and transferred to nitrocellulose. Blots were incubated with 10⁷ PFU/ml of LCMV cl-13. Bound virus was detected with anti-LCMVGP MAbs 33.1 and 36.1 and by ECL. (D) Solid-phase virus binding assay. Equal amounts of purified DGFc1 through -5 were immobilized in microtiter plates and incubated with 10⁷ PFU/ml LCMV cl-13. Bound virus was detected with anti-LCMVGP2 MAb 83.6 and an HRP-conjugated secondary antibody using ABTS substrate. Optical density at 405 nm [OD (405)] was recorded in an ELISA reader and background binding to BSA subtracted (mean ± standard deviation; n = 3). (E) Binding of virus and laminin to DGFc mutants. Serial dilutions of equal amounts of purified DGFc5R312K, DGFc5R312A, and DGFc5 were subjected to Western blot analysis and VOPBA as described for panels B and C. For laminin overlay, 10 μg/ml EHS laminin-1 was used and bound laminin detected with a polyclonal anti-laminin-1 antibody using ECL. The prominent band at the bottom, close to the running front of the gel, was observed in some preparations of DGFc5 and may represent degradation products. wt, wild type. (F) Binding of DGFc5R312A and DGFc5 to virus. LCMV cl-13 was immobilized in microtiter plates and incubated with purified DGFc5R312A and DGFc5, which were detected with an HRP-conjugated anti-human Fc antibody in a color reaction using ABTS substrate (mean ± standard deviation; n = 3).

FIG. 2.

Virus binding to α-DG depends on posttranslational modification. (A) Schematic representation of the DGFc5 deletion mutants. Domains of DG are depicted as described in the legend to Fig. 1. Equal amounts of DGFc5 deletion mutants and wild-type (wt) DGFc5 purified from the supernatants of transiently transfected HEK293T cells were subjected to Western blot analysis with an anti-Fc antibody (B) as described in the legend to Fig. 1B. The HA epitope present in DGFc5Δ30-316 was detected with the anti-HA antibody Y11 (C). VOPBA with LCMV cl-13 (D) and the solid-phase virus binding assay (E) were performed as described in the legend to Fig. 1C and 1D, respectively. OD (405), optical density at 405 nm. LOA (F) was done as described in the legend to Fig. 1F. For the solid-phase laminin binding assay (G) immobilized DGFc5 variants were incubated with 10 μg/ml laminin-1, which was then detected with an anti-laminin-1 antibody, using an HRP-conjugated secondary antibody (mean ± standard deviation; n = 3). SP, signal peptide.

FIG. 3.

Virus binding to α-DG increases after LARGE overexpression. (A) Binding of virus and laminin to DGFc5 from cells overexpressing LARGE. HEK293T cells transiently transfected with DGFc5 were infected with Ad5/LARGE-EGFP (LARGE) or Ad5/EGFP (EGFP). After 48 h, DGFc5 was purified from culture supernatants. Serial dilutions of equal amounts of DGFc5 were subjected to Western blot analysis using an anti-Fc antibody, LOA, and VOPBA as described in the legend to Fig. 1. (B) Binding of DGFc5 from cells overexpressing LARGE to virus. Immobilized LCMV cl-13 was incubated with the indicated concentrations of DGFc5 purified from cells overexpressing LARGE or EGFP controls and bound DGFc5 detected as described in the legend to Fig. 1F. OD (405), optical density at 405 nm. (C) DGFc5Δ30-168 is recognized by LARGE. Equal amounts of DGFc5Δ30-168 (Δ30-168) and wild-type DGFc5 (wt) purified from cells overexpressing LARGE (L) or EGFP controls (E) were subjected to Western blot analysis, LOA, and VOPBA as described for panel A.

FIG. 4.

Monoclonal antibody IIH6 blocks virus-receptor interaction. (A) Blocking of LCMV/α-DG binding by MAb IIH6. DGFc5, α-DG from DG^+/− ES cells, MC57 cells (MC57), or rabbit skeletal muscle (SM) was immobilized on nitrocellulose and preincubated with the indicated concentrations of MAb IIH6. Blots were then incubated with 10⁷ PFU/ml of LCMV cl-13 in the presence of the same concentrations of MAb IIH6. After several washes, bound virus was detected with anti-LCMVGP MAbs 33.1 and 36.1 and ECL. (B) Neutralization of LCMV infection with soluble α-DG. LCMV cl-13 (200 PFU) was incubated with the indicated concentrations of α-DG or DGFc1 for 1 h on ice and then added to either DG^+/− ES cells (filled symbols) or DG^−/− ES cells (open symbols). LCMV infection was assessed after 16 h by immunofluorescence staining for LCMVNP. (mean ± standard deviation; n = 3). (C) Blocking of infection with MAb IIH6. DG^+/− ES cells were blocked with MAb IIH6 or an unrelated mouse IgM (control) for 2 h at 4°C. Next, 200 PFU of LCMV cl-13 was added for 45 min and infection assessed as described for panel B.

FIG. 5.

Tissue-specific LARGE-dependent modification of α-DG correlates with virus binding affinity. Equal amounts of total tissue homogenates of adult mouse skeletal muscle (lane 1), liver (lane 2), kidney (lane 3), brain (lane 4), heart (lane 5), and lung (lane 6) were subjected to WGA affinity chromatography. Eluted glycoproteins were probed with an antibody recognizing the core protein of α-DG and MAb IIH6 in Western blot analysis. LOA and VOPBA were performed as described in the legend to Fig. 1. Molecular masses are indicated to the left of the gels.

FIG. 6.

α-DG derived from LARGE^myd mice shows reduced binding to laminin and virus. α-DG was isolated from the skeletal muscle (skm), brain, and liver of homozygous LARGE^myd mice (m) and control littermates (wt) by WGA affinity purification and was probed with the antibody FPD and MAb IIH6 in Western blot analysis, LOA, and VOPBA as described in the legend to Fig. 5.

FIG. 7.

LARGE-mediated modification of α-DG is critical for the recognition by different LCMV isolates. (A) Virus binding to DGFc5 deletion mutants. Immobilized DGFc5 variants were incubated with LCMV cl-13 (10⁷ PFU/ml) and ARM53b (10⁸ PFU/ml). For the detection of bound viruses, MAb 83.6 was combined with a biotinylated anti-mouse IgG secondary antibody and HRP-streptavidin (mean ± standard deviation; n = 3). OD (405), optical density at 405 nm. (B) Binding of DGFc5Δ30-312 and DGFc5 to LCMV isolates. Equal amounts of purified viruses were immobilized and incubated with the indicated concentrations of DGFc5Δ30-312 (filled symbols) and DGFc5 (open symbols). Bound DGFc5 was detected using a mouse anti-human Fc antibody, combined with a biotinylated anti-mouse IgG and HRP-streptavidin (mean ± standard deviation; n = 3). (C) Binding of DGFc5 from cells overexpressing LARGE to LCMV isolates. Immobilized viruses were incubated with DGFc5 from cells overexpressing LARGE (filled symbols) or EGFP (open symbols). Bound DGFc5 was detected as described for panel B (mean ± standard deviation; n = 3). wt, wild type.

FIG. 8.

Modification of α-DG by LARGE is involved in binding of LFV, Mobala, and Oliveros. Binding of LFV, Mobala, Oliveros, and Guanarito to DGFc5 deletion mutants (A) and DGFc5 derived from cells overexpressing LARGE or EGFP (B). Immobilized DGFc5 variants were incubated with γ-inactivated LFV, Mobala, Oliveros, and Guanarito (10⁷ PFU/ml). Bound viruses were detected with MAbs 83.6 and 33.1 using an HRP-conjugated secondary antibody and ECL. Exposure times were 1 min for LFV, Mobala, and Oliveros and 10 min for Guanarito. LOA and Western blot analysis with anti-Fc antibody were performed as described in the legend to Fig. 3. (C) Binding of DGFc5 variants to immobilized viruses. Equal amounts of purified viruses were immobilized and incubated with the indicated concentrations of DGFc5Δ30-312, DGFc5, and DGFc5 from cells overexpressing EGFP (DGFc5 EGFP) or LARGE (DGFc5 LARGE). OD (405), optical density at 405 nm. Bound DGFc5 was detected as described in the legend to Fig. 7C (mean ± standard deviation; n = 3). wt, wild type.

FIG. 9.

LARGE-dependent modification is crucial for α-DG's ability to mediate infection by several isolates of LCMV and recombinant VSV pseudotyped with LFVGP. (A) Schematic representation of wild-type DG and the DG deletion mutant DGΔ30-316. The putative N-terminal subdomains (white), the mucin-type domain (black), and the C-terminal globular domain (gray) of α-DG are indicated. Amino acids 653 through 895 represent β-DG with the transmembrane domain (dark box). The influenza HA epitope in DGΔ30-316 is indicated. SP, signal peptide. (B) Expression of wild-type DG and DGΔ30-316. DG^−/− ES cells were infected with AdV vectors containing DGΔ30-316 (lane 1), wild-type DG (lane 2), or a β-galactosidase reporter gene (LacZ) (lane 3) at an MOI of 10. Lane 4 represents the parental DG^+/− ES cells in parallel. After 48 h, cells were lysed and total protein isolated, separated by SDS-PAGE, and transferred to nitrocellulose. Blots were probed with anti-β-DG polyclonal antibody AP83, anti-HA antibody Y11, and anti-α-DG MAb IIH6. Primary antibodies were detected with HRP-conjugated secondary antibodies and by ECL. (C and D) Reconstitution of virus infection in DG^−/− ES cells by wild-type (wt) DG and DGΔ30-316. DG^−/− ES cells were infected with AdV vectors containing DGΔ30-316 (white), wild-type DG (light gray), or LacZ (dark gray). As a positive control, DG^+/− ES cells were cultured in parallel (black). After 48 h, cells were infected with 200 infectious units/well of either the LCMV variants indicated (C) or the VSV pseudotypes VSVΔG*-LFVGP (LFV) or VSVΔG*-VSVGP (VSV). Infection levels in panel C were assessed by immunofluorescence staining for LCMVNP as described in the legend to Fig. 4B. Data in panel D are for EGFP-positive cells per well (mean ± standard deviation; n = 3).

FIG. 10.

Modification of α-DG by LARGE influences the competition between virus and laminin for receptor binding. Equal amounts of DGFc5 produced in cells overexpressing LARGE (DGFc5 LARGE) or EGFP (DGFc5 EGFP) were immobilized in microtiter plates and preincubated with the indicated concentrations of laminin-1 (filled symbols) or fibronectin (open symbols). LCMV cl-13 (10⁷ PFU/ml) and ARM53b (10⁸ PFU/ml) were added and bound virus detected as described in the legend to Fig. 7A (mean ± standard deviation; n = 3).OD (405), optical density at 405 nm.

FIG. 11.

Modification of α-DG by LARGE influences the ability of the virus to compete with cell-associated laminin. (A) Overexpression of LARGE in Vero cells. Vero cells were infected with Ad5/LARGE-EGFP and Ad5/EGFP (MOI = 10). After 24 h, cells were fixed and LARGE-modified α-DG was detected using MAb IIH6 and a rhodamine-X (red)-coupled anti-mouse IgM. EGFP was detected by direct fluorescence. Bar, 20 μm. (B) laminin and virus binding to α-DG. Vero cells were infected with Ad5/LARGE-EGFP and Ad5/EGFP at MOIs of 0, 10, and 100. After 48 h, cells were lysed and α-DG extracted and examined by LOA and VOPBA as described in the legend to Fig. 3. (C and D) Blocking of virus infection with laminin-1. Vero cells overexpressing LARGE or EGFP were cultivated on laminin in the absence (0) or presence of 5, 20, and 50 μg/ml soluble laminin-1 (black bars) or fibronectin (white bars) for 12 h. Cells were then infected with either 200 PFU LCMV (C) or VSVΔG* pseudotypes (D) for 16 h. Infection levels in panel C were assessed by immunofluorescence staining for LCMVNP as described in the legend to Fig. 4B. Data in panel D are for EGFP-positive cells per well (mean ± standard deviation; n = 3).