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Identification of α-Dystroglycan as a Receptor for Lymphocytic Choriomeningitis Virus and Lassa Fever Virus

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Science  11 Dec 1998:
Vol. 282, Issue 5396, pp. 2079-2081
DOI: 10.1126/science.282.5396.2079

Abstract

A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be α-dystroglycan (α-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified α-DG protein. Soluble α-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, α-DG is a cellular receptor for both LCMV and LFV.

Arenaviruses consist of several causative agents of fatal human hemorrhagic fevers (1, 2). Among these pathogens, LFV causes an estimated 250,000 cases and more than 5000 deaths annually (1,3). LCMV, the prototype arenavirus, has been studied primarily in its natural rodent host as a model of viral immunology and pathogenesis (4).

To initiate infection, the LCMV glycoprotein GP-1 anchors the virus to the cell surface through a proteinaceous receptor (5, 6), which by a virus overlay protein blot assay (VOPBA) (7) was identified as a single high molecular weight glycoprotein (5). The presence of the receptor protein correlated directly with a cell's susceptibility to LCMV attachment and infection (Fig. 1). Its broad migration pattern on SDS-polyacrylamide gels is likely to reflect the heterogeneity in cell type–specific posttranslational modifications (5). In addition to murine cells, a broad range of rodent and primate cells express the same protein (5) (Fig. 1). LFV bound to what appears to be the same glycoprotein (Fig. 1), suggesting that both viruses may share a common cellular receptor.

Figure 1

Binding of LCMV and LFV to cell membrane proteins. VOPBA was performed with either enriched uninfected baby hamster kidney cell culture fluid (Mock), LCMV (strain Cl 13), or LFV on identical blots. Each lane on the blot contained 50 μg of purified cell membrane proteins prepared from two cell lines susceptible to LCMV infection: monkey kidney line Vero E6 (lane 1) and mouse fibroblast line MC57 (lane 3); and two cell lines resistant to LCMV infection and binding, human T lymphocyte line Jurkat (lane 2) and mouse T lymphocyte line RMA (lane 4). Molecular size markers are indicated at the sides (in kilodaltons).

This putative receptor protein was purified from a LCMV permissive cell line by sequential column chromatography (8), and a total of five tryptic peptides were sequenced (9). Peptides GT384, GT441, and GT417 showed complete homology to dystroglycan (DG) precursor protein at the regions of amino acids 610 to 623, 571 to 585, 516 to 533, respectively. DG is encoded by a single gene and processed into two mature proteins, α- and β-DG, which form a complex spanning the plasma membrane (10). The sequences corresponding to the three peptides are near the COOH-terminus of α-DG. α-DG is an extracellular peripheral membrane protein that binds to the extracellular matrix and is noncovalently associated with β-DG, which is a transmembrane protein linked to the cytoskeleton (11). DG complex is expressed in a wide variety of tissues and cells, and plays an important role in mediating cell-extracellular matrix interactions (12, 13). The LCMV receptor was preferentially expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells, facilitating a basolateral route of LCMV entry (14). This parallels a similar localization of DG expression (12).

The interaction between α-DG and LCMV was demonstrated in VOPBAs with several strains of LCMV [Cl 13, Armstrong 5 (Arm5), and WE54] on blots containing purified α-DG proteins (Fig. 2B). All these LCMV strains bound to purified native α-DG protein (15) (Fig. 2B). In contrast, none of these viruses recognized the Escherichia coli– expressed glutathione S-transferase (GST)– fusion proteins with either FP-D or FP-B (Fig. 2B), which encode different regions of DG precursor sequence (10) (Fig. 2A), suggesting that the extreme NH2-terminus of α-DG and possibly posttranslational modifications on this protein are crucial for LCMV recognition. A similarly purified glycoprotein, the α2 subunit of the dihydropyridine receptor complex (16), also negatively charged through glycosylation, did not bind to LCMV (17). Like LCMV, the arenaviruses LFV, Mobala, and Oliveros bound to purified native α-DG protein, but not to the recombinant GST–FP-D or GST–FP-B proteins (Fig. 2C). In contrast, the arenavirus Guanarito failed to recognize α-DG protein (Fig. 2C).

Figure 2

Binding of LCMV, LFV, and other arenaviruses to α-DG. (A) Diagram showing DG precursor and GST-fusion proteins. The signal sequence (residues 1 to 28) is shown as a solid box. The sequence corresponding to α-DG (residues 29 to 653) and β-DG (residues 654 to 895) are shown as open boxes. Fusion protein GST–FP-D contains residues 62 to 438 of the DG precursor. Fusion protein GST–FP-B contains residues 367 to 863 of the DG precursor. (B) Four identical blots containing purified DG were used in VOPBAs with either no virus (nil) or LCMV strains Cl 13, Arm5, and WE54. Each blot contained protein samples from three different sources. Lane A contained 10 μg of purified 156-kD α-DG protein from rabbit skeletal muscle; lane B contained 40 μg of recombinant 68-kD GST–FP-D; and lane C contained 40 μg of recombinant 82-kD GST–FP-B. Arrows indicate the expected full-length proteins. (C) Blots as described in (B) were used in VOPBA with arenaviruses LFV, Mobala, Oliveros, and Guanarito.

Purified soluble α-DG competed with the cell surface virus receptor during LCMV infections in a dose-dependent manner (Fig. 3). α-DG at 0.9 nM concentration effectively blocked LCMV infectivity (18) (Fig. 3A) and significantly reduced production of progeny LCMV (Fig. 3B). Soluble α-DG blocked infection by LFV at similar concentrations, but roughly 100-fold higher amounts were required to block LCMV Arm5 infectivity (17). In contrast, α-DG had no effect on the infection by an unrelated RNA virus vesicular stomatitis virus (VSV) (Fig. 3B).

Figure 3

Blocking of infection by LCMV with soluble α-DG protein. (A) 3T6 mouse fibroblast cells were infected with LCMV strain Cl 13 or VSV in the presence of increasing concentrations of α-DG. As a control, LCMV Cl 13 infection was also performed in the presence of increasing concentrations of bovine serum albumin. Sixteen hours later, cells were immunostained with specific antibodies to detect LCMV nucleoprotein or VSV glycoprotein (18), and the number of infected cells was observed under fluorescence microscopy. The results were quantitated as an average of at least four fluorescent areas and plotted as percentage of control where no competing protein was added. (B) Virus titers in the 3T6 culture supernatants 16 hours after infection were determined by plaque assay. Virus titers [log (plaque-forming units) per milliliter] are plotted against the concentration of competing proteins added during absorption.

A mouse embryonic stem (ES) cell line expressing DG [wild type (wt)] was readily infected by LCMV (Fig. 4; at a MOI of 10, >90% of cells were infected). However, LCMV replication did not occur in DG null ES (ko) cells (19) (Fig. 4; at a MOI of 10, <0.1% of cells were infected), yet these cells were viable, maintained a growth rate similar to the parental cells, and were equally infectable by VSV (17). When the α-DG protein was restored on the surface of ko cells by infection with an adenovirus vector carrying DG cDNA (19), these cells again became susceptible to LCMV infection (20) (Fig. 4; at a MOI of 10, ∼75% of cells were infected). However, when the same adenovirus vector expressing green fluorescent protein or LacZ was used for infection, less than 0.1% of ko cells were infectable by LCMV (17).

Figure 4

Requirement of α-DG expression by LCMV infection. Parental wt ES cells, DG null ko cells, and ko cells reconstituted with dystroglycan (ko+DG) were infected with LCMV strain Cl 13 at a multiplicity of infection (MOI) of 0.1, 1, and 10, respectively. Sixteen hours later, cells were stained with mAb 1-1-3 to detect LCMV nucleoprotein by immunofluorescence. DG reconstitution was achieved by infecting ko cells with an adenovirus vector carrying rabbit DG cDNA.

Old World arenaviruses including LCMV, LFV, and Mobala, which are phylogenetically and serologically distinct from the New World arenaviruses (1, 21), specifically recognized the α-DG receptor protein. Oliveros virus, a group C New World arenavirus (1, 22), also bound to α-DG in VOPBA, but Guanarito, another New World arenavirus, failed to do so. Use of ES ko cells alone and reconstituted with DG may prove valuable to further group the arenaviruses.

Cellular receptors are key elements in determining the tropism and pathogenesis of virus infection. The presence of α-DG in all the tissues and organs examined to date correlates with the tropism of LCMV (5, 23). High sequence conservation in the gene encoding DG (11) supports the broad host range for LCMV and arenavirus infections of animals and humans (1). Characterization of the α-DG–arenavirus interaction should elucidate the early events of arenavirus infection and facilitate the development of strategies to intervene and prevent this crucial interaction.

  • * To whom correspondence should be addressed. E-mail address: mbaobo{at}scripps.edu

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