Characterization of the interaction of lassa fever virus with its cellular receptor alpha-dystroglycan

Abstract

The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as alpha-dystroglycan (alpha-DG), a cell surface receptor that provides a molecular link between the extracellular matrix and the actin-based cytoskeleton. In the present study, we show that LFV binds to alpha-DG with high affinity in the low-nanomolar range. Recombinant vesicular stomatitis virus pseudotyped with LFV glycoprotein (GP) adopted the receptor binding characteristics of LFV and depended on alpha-DG for infection of cells. Mapping of the binding site of LFV on alpha-DG revealed that LFV binding required the same domains of alpha-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin alpha1 and alpha2 chains for alpha-DG binding. Together with our previous studies on receptor binding of the prototypic immunosuppressive LCMV isolate LCMV clone 13, these findings indicate a high degree of conservation in the receptor binding characteristics between the highly human-pathogenic LFV and murine-immunosuppressive LCMV isolates.

FIG. 1.

LFV binds α-DG with high affinity. (A) Detection of viral GPs in purified LFV and LCMV. Purified LFV strain Josiah (inactivated by γ-irradiation) and LCMV cl-13 (10⁷ PFU/ml each) were solubilized in SDS-PAGE buffer, and 40 μl (lanes 1) and 10 μl (lanes 2) of the samples were separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with MAb 83.6 anti-LCMVGP2, using a peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL) for detection. Molecular masses are indicated. (B) Comparison of the binding affinities for α-DG of LFV and LCMV cl-13 by VOPBA. α-DG, purified from rabbit skeletal muscle, was diluted (1, 0.1, 0.01, 0.001 μg) and blotted to nitrocellulose. LFV and LCMV cl-13 were applied at the same concentration (10⁷ PFU/ml). Bound virus was detected by monoclonal antibodies 33.6 and 86.6 that recognize conserved epitopes of LFV and LCMV GP2, using a peroxidase-conjugated anti-mouse IgG secondary antibody and enhanced chemiluminescence (ECL). (C) Binding of α-DG to LFV, LCMV cl-13, and LCMV WE2.2. LFV Josiah (circles), LCMV cl-13 (squares), and LCMV WE2.2 (triangles) were immobilized in a microtiter plate and incubated with biotinylated α-DG. Bound biotinylated or α-DG was detected with peroxidase-conjugated streptavidin in a color reaction using ABTS substrate. OD₄₀₅ was recorded in an ELISA reader. For the determination of specific binding, background binding to BSA was subtracted (mean ± standard deviation; n = 3).

FIG. 2.

Characterization of recombinant LFVGP. (A) Expression of recombinant LFVGP. HeLa cells were transfected with the expression construct pC-LFVGP (lane 1), pC-EGFP (lane 2), and empty vector (lane 3) using Lipofectamine. Two days after transfection, cells were lysed in SDS-PAGE sample buffer. As a positive control, a lysate of purified LFV virions (10⁷ PFU/ml) was used (lane 4). Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using MAb 83.6 anti-LCMVGP2. Bound primary antibody was detected with a peroxidase-conjugated secondary antibody using ECL for detection. Molecular masses are indicated. (B) Cell surface labeling of recombinant LFVGP. HeLa cells were transfected with pC-LFVGP (LFVGP) or empty vector (mock) as in panel A. Cell surface LFVGP was detected with MAb 83.6 anti-LCMVGP2 and a PE-conjugated secondary antibody to mouse IgG. For flow cytometry, a FACSCalibur flow cytometer was used. In histograms, the y axis represents cell numbers and the x axis represents fluorescence intensity (FL2-H for anti-mouse IgG-PE conjugate).

FIG. 3.

Recombinant vesicular stomatitis virus pseudotyped with LFVGP adopts the receptor binding characteristics of LFV and depends on α-DG for infection. (A) Blocking of VSVΔG*-LFVGP infection by γ-inactivated LFV. HeLa cells cultured in 96-well plates were blocked with the indicated PFU/cell of γ-inactivated LFV or γ-inactivated Amapari. After incubation for two hours at 4°C, cells were infected with 200 IU of VSVΔG*-LFVGP (black bars) or VSVΔG*-VSVGP (white bars). Infection was assessed after 24 h by detection of the EGFP reporter in fluorescence microscopy. Data are EGFP-positive cells per well (n = 3; ± standard deviation). (B) Infection with LFV-PS depends on α-DG. DG^−/− ES cells were infected with AdV vectors containing either wild-type DG or the deletion mutant DGH at an MOI of 10. After 48 h, AdV-infected cells as well as DG^−/− and DG^+/− ES cells cultured in parallel were infected with 200 IU of VSVΔG*-LFVGP or VSVΔG*-VSVGP and infection was assessed after 24 h as in panel B (n = 6; ± standard deviation).

FIG. 4.

Structures within amino acids 169 to 408 are required for LFV binding. (A) Schematic representation of the α-DG deletion mutants: 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 to 895 represent β-DG with the transmembrane domain (dark box). The influenza virus hemagglutinin (HA) epitope in DGE is indicated. (B) Expression of the α-DG deletion mutants. DG^−/− ES cells were infected with AdV vectors containing the α-DG variants or green fluorescent protein (−). After 48 h, cells were lysed and samples subjected to jacalin affinity chromatography. Jacalin-bound glycoproteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-β-DG. wt, wild type. (C) Binding of LCMV cl-13 and LFV to wild-type and deletion mutants of α-DG. Jacalin-bound glycoproteins (B) were subjected to VOPBA with 10⁷ PFU/ml of LFV using MAb 83.6, anti-LCMVGP2, and enhanced chemiluminescence (ECL) for detection. (D) Reconstitution of LFV-PS infectivity in DG^−/− ES cells by wild-type and deletion mutants of α-DG. DG^−/− ES cells were infected with AdV vectors containing wild-type and deletion mutants of α-DG or a β-galactosidase reporter gene (LacZ) at an MOI of 10. After 48 h, transgene expression was verified by detection of β-DG in total cell protein in the Western blot. Cells cultured in parallel were infected with 200 IU (MOI = 0.01) of VSVΔG*-LFVGP (black bars) or VSVΔG*-VSVGP (white bars), and infection was assessed after 24 h as in panel B. Infection levels were assessed 24 h later by the detection of GFP-positive cells by immunofluorescence (n = 6; ± standard deviation).

FIG. 5.

Amino acids 169 to 408 of α-DG contain a high-affinity binding site for LFV and LCMV. (A) Schematic representation of the α-DG fragments fused to human IgG Fc. The putative domains of α-DG are depicted as in Fig. 4, and human IgG1Fc is indicated. wt, wild type. (B) Purified α-DG-Fc fusion proteins. The α-DG-Fc fusion proteins DGFc1 through -5 were expressed in HEK293T cells and purified by protein A affinity chromatography from cell lysates. Purified proteins were analyzed by SDS-PAGE with Coomassie blue staining (B) and by Western blot using an anti-human IgG Fc antibody (C). (D) Binding of LFV to the α-DG-Fc fusion proteins. Equal amounts of purified DGFc1 through -5 were immobilized in microtiter plates and incubated with 10⁷ PFU/ml γ-inactivated LFV. Bound virus was detected with MAb 83.6 (anti-LCMVGP2) and a peroxidase-conjugated secondary antibody in a color reaction using ABTS substrate. OD₄₀₅ was recorded in an ELISA reader. For the determination of specific binding, background binding to BSA was subtracted (mean ± standard deviation; n = 3).

FIG. 6.

LFV and LCMV cl-13 compete with laminin α1 and α2 chains for α-DG binding. (A) Blocking of virus binding to α-DG by laminin-1 and laminin-2. α-DG purified from rabbit skeletal muscle was immobilized in microtiter plates and blocked with the indicated concentrations of laminin-1 (black), laminin-2 (gray), and fibronectin (white) for 4 h at room temperature. Inactivated LFV and LCMV cl-13 were added in a dilution of 10⁷ PFU/ml containing the blocking proteins in the concentrations mentioned above for 12 h at 6°C. Bound virus was detected as in Fig. 5D. For the determination of specific binding, background binding to BSA was subtracted (mean ± standard deviation; n = 3). (B) Displacement of LFV bound to α-DG with soluble laminin-1, laminin-2, and fibronectin: Inactivated LFV and LCMV cl-13 (10⁷ PFU/ml) were bound to immobilized α-DG. Plates were washed, and the indicated concentrations of ECM proteins were then added for 4 h at room temperature. After three wash steps, bound virus was detected as described in panel A.