Article |
Address correspondence to Michael B.A. Oldstone, The Scripps Research Institute, Div. of Virology, Dept. of Neuropharmacology, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-8054. Fax: (858) 784-9981. E-mail: mbaobo{at}scripps.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: lymphocytic choriomeningitis virus; viral receptor; binding site; extracellular matrix; pathogenesis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DG is a highly versatile cell surface receptor that provides a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton (Ervasti and Campbell, 1991, 1993; Ibraghimov-Beskrovnaya et al., 1992; Henry and Campbell, 1999; Winder, 2001). Initially encoded as a single protein, DG undergoes posttranslational cleavage to form -DG, a peripheral protein, and ß-DG, a membrane protein. DG is expressed in most developing and adult tissues, typically in cell types that adjoin basement membranes (Durbeej et al., 1998). At those sites, DG plays a critical role in cell-mediated assembly of basement membranes (Henry and Campbell, 1998).
-DG undergoes high affinity interactions with the ECM proteins laminin-1, laminin-2, agrin, and perlecan (Ervasti and Campbell, 1991, 1993; Gee et al., 1994; Talts et al., 1999). Biochemical and structural data suggest that these interactions engage a lectin-type binding involving
-DGderived carbohydrates (Ervasti and Campbell, 1993; Pall et al., 1996; Yamada et al., 1996; Hohenester et al., 1999).
-DG is non covalently associated with the membrane-spanning ß-DG, which binds to the cytoskeletal proteins dystrophin and utrophin, and the signal transduction molecules grb2 and focal adhesion kinase (Henry and Campbell, 1999). The complex binding pattern of
-DG likely results in the formation of tissue-specific
-DG complexes in the cell membrane. Since specific DG-associated proteins may enhance or block virus binding, their coexpression with DG may modulate the susceptibility of a specific cell for virus infection and represent an important determinant for viral tissue tropism in vivo. Based on their ubiquitous expression, high binding affinity, and large size, ECM molecules may interfere with virus binding to
-DG and therefore provide a barrier for virus infection in vivo. Clearly, the ability of a virus to displace those ECM proteins in order to bind its cellular receptor would be determined by its receptor binding affinity.
Recent studies from our laboratory have documented a striking and consistent correlation between F260L or F260I mutations in LCMV-GP1 and the affinity of LCMV binding to -DG, tissue tropism, and pathological potential (Borrow et al., 1995; Sevilla et al., 2000; Smelt et al., 2001). LCMV strains and variants with high binding affinity to
-DG replicate preferentially in the white pulp of the spleen and infect
-DG+/CD11c+ dendritic cells (DC). Infection of this important population of antigen-presenting cells results in a generalized immunosuppression of the host. In contrast, strains and variants with low binding affinity for
-DG localize primarily to the red pulp of the spleen, show only very limited infection of CD11c+ DC and do not cause immunosuppression. Understanding this pivotal linkage between receptor binding and viral pathogenesis requires detailed molecular analysis of the virus
-DG interaction.
The present study characterizes LCMV-DG binding biochemically and analyzes the role of
-DGassociated host-derived proteins in the virus infection. The results emphasize that LCMV
-DG binding is a novel type of interaction involving globular protein domains and mucin-related structures of
-DG. Despite several defined biochemical differences in binding, our data indicate that LCMV and laminin-1 use overlapping binding sites on
-DG and that the ability of a LCMV isolate to compete with laminin-1 for receptor binding is determined by the virus affinity for
-DG.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Next, we produced soluble -DG fragments as transcriptional fusion proteins with the heavy chain constant region of human IgG1 (IgG1 Fc) containing the
-DG sequences 30181 (DGFc1), 30316 (DGFc2), 30408 (DGFc3), 30485 (DGFc4), and full-length
-DG 30653 (DGFc5) (Fig. 5 A).
-DGFc fusion proteins were expressed in HEK293T cells and purified by protein A affinity chromatography. The molecular masses of the recombinant
-DGFc fusion proteins were in agreement with the expected values, and extensive dimerization of the
-DGFc fusion proteins was observed under nonreducing conditions. Equal amounts of the purified proteins DGFc1 through 5 were analyzed by immunoblotting using a polyclonal antihuman IgGFc antibody (Fig. 5 B). VOPBA with LCMV clone-13 revealed virus binding to DGFc3 through 5 but not to the smaller fragments DGFc1 and DGFc2 (Fig. 5 C). To test virus binding to the
-DGFc fusion proteins under nondenaturing conditions, a dot-VOPBA was performed. Despite comparable amounts of protein immobilized, as assessed by detection of human IgG1 Fc, only DGFc3 and DGFc4 but not DGFc1 or DGFc2 exhibited virus binding (Fig. 5 D). These results combined with those from deletion mutagenesis suggest that amino acids 169408 contain structures necessary and sufficient for the LCMV receptor function of
-DG.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biochemical characterization suggests proteinprotein contacts rather than a lectin-type binding in the LCMV-DG interaction
Considerable evidence suggests that -DG ECM protein interactions require
-DGderived carbohydrate structures. Further, the absolute dependence of
-DG ECM protein interactions on divalent cations, the extreme sensitivity to chelating agents, enhanced salt concentration, and in some cases low concentration of heparin (Ervasti and Campbell, 1993; Pall et al., 1996, Andac et al., 1999), all coincide with the recently solved structure of the
-DG binding sites on laminin-2 (Hohenester et al., 1999). In contrast, our results indicate that LCMV
-DG binding is independent of divalent cations and insensitive to chelating agents and heparin. Extensive neuraminidase treatment, oxidation by low concentrations of sodium periodate, and competition with soluble sialic acid, which can reduce the laminin-1
-DG interaction (Yamada et al., 1996; Rambukkana et al., 1998), had no effect on virus binding. Our data agree with previous studies demonstrating LCMV binding to cells lacking N- and O-linked sugars after treatment with N- and O-glycanase or virus binding to cells incubated overnight with tunicamycin, which inhibits N-linked glycosylation (Borrow and Oldstone, 1992). Here, we noted that only extensive deglycosylation of
-DG reduced virus binding, likely reflecting changes in the overall conformation of the protein rather than a direct involvement of carbohydrates. Further, the dramatic changes in virus binding by the single point mutations F260L (ARH53b/clone-13) and S153F (WE54/WE22) in the LCMV-GP1 (Sevilla et al., 2000) suggest that proteinprotein interactions rather than a carbohydrate-mediated lectin-type binding play a crucial role in the formation of viral GP1
-DG complexes. However, the marked reduction of virus binding upon deglycosylation denotes an important function of carbohydrates for the conformational stability and/or proper folding of the virus binding site. This indirect role of
-DGderived glycans is supported by our finding that structures within the mucin-related domain of
-DG are necessary for virus binding.
Virus binding involves the COOH-terminal part of the NH2-terminal globular domain and the NH2-terminal part of the mucin-related central domain of -DG
Using a combination of deletion mutagenesis and binding studies with -DG fragments, we found that structures formed by amino acids 169408 of
-DG are necessary and sufficient for virus binding. Based on its amino acid sequence, a modular structure of
-DG has been proposed (Ibraghimov-Beskrovnaya et al., 1992). Presumably, amino acids 30316 of
-DG form an independent folding unit composed of at least two independent subdomains, that is, residues 30180 and residues 181316 (Brancaccio et al., 1997). The expression of the DG mutants DGI (
H30-A168), DGD (
A182-315), and DGE (
H30-A316) as stable proteins in eukaryotic cells supports this proposed modular structure. The central region of
-DG (residues 316485) is related to mucins and contains a great number of terminally sialylated O-linked sugar chains that exhibit remarkable tissue-specific differences. Based on these data, the virus-binding region mapped in this study included the proposed second globular subdomain of the NH2-terminal portion of
-DG and part of the adjacent mucin-related domain. Our biochemical data dispute a participation of terminal sialic acids of the mucin-type O-linked sugar chains as proposed by some studies for laminin binding (Yamada et al., 1996), making a function of mucin-type glycans for the conformational stability and/or proper folding of the virus binding site more probable. However, binding to the polypeptide core structure of the mucin-type domain or recognition of some unknown structures such as specific carbohydrate entities or chemically modified amino acid side chains by the virus remains possible.
The binding site of LCMV on -DG overlaps with the one used by laminin-1 and likely other ECM molecules
A striking feature of DG is its complex binding pattern involving a wide variety of proteins. Hence, -DG at the cell surface might be covered by ECM molecules and cellular proteins. Some of these
-DGassociated proteins may enhance virus binding, perhaps as cofactors or coreceptors. Other DG-associated proteins may compete with the virus for overlapping binding sites on
-DG and thereby block its function as a viral receptor. Using purified proteins in an in vitro binding assay, we demonstrated a competition between laminin-1 and LCMV for
-DG binding. Since laminin-1, laminin-2, agrin, and perlecan occupy overlapping binding sites on
-DG (Talts et al., 1999), our data for laminin-1 likely apply to these ECM components. Therefore, the susceptibility of a specific cell to arenavirus infection in vivo may depend not only on the expression of
-DG itself but also on
-DG's association with host-derived proteins. If so, binding affinity for
-DG represents a crucial determinant for the tissue tropism and selection of viruses within a given host (Borrow et al., 1995; Sevilla et al., 2000).
Competition between virus and ECM molecules for -DG likely underlies the observation that receptor binding affinity is a crucial determinant for tissue tropism and subsequent pathogenesis of selected LCMV strains and variants
Comparative studies in vivo revealed that the closely related LCMV ARM53b and clone-13 display markedly different courses of disease. Both viruses cause a persistent infection upon congenital, neonatal, or in utero infection. However, infection of adult immunocompetent animals only with clone-13 but not ARM53b causes a persistent infection (Ahmed et al., 1984). In contrast to the persistent infection initiated early in life that is due to thymic deletion of antiviral immune cells, persistent infection of adult animals with clone-13 is associated with a generalized immunosuppression (Oldstone et al., 1973; Borrow et al., 1995). These distinct pathologies of ARM53b and clone-13 are reflected by a different tissue tropism in spleen. Clone-13 was noted to replicate preferentially in the white pulp of the spleen and infect CD11c+ interdigitating DC, whereas in contrast ARM53b localized primarily to the red pulp of the spleen with almost complete absence from the white pulp (Borrow et al., 1995). At the structural level, clone-13 differs from ARM53b by only two point mutations, K1079Q in the viral polymerase and F260L in the viral glycoprotein (Salvato et al., 1988; Salvato et al., 1991). Although both viruses use -DG as their cellular receptor (Cao et al., 1998), clone-13 binds to
-DG with much higher affinity than ARM53b (Sevilla et al., 2000). Recent comprehensive studies of numerous LCMV strains and variants generated in vivo demonstrated that enhanced receptor binding affinity, tropism to DC, biological consequences of immunosuppression, and persistent infection all reflect consistently a F260L or F260I mutation in LCMV-GP1, the component that binds the cellular receptor (Sevilla et al., 2000). The selection of LCMV variants over time in different lymphoid and other organs further reveals strong selective pressure for an aliphatic amino acid in position 260 of the LCMV-GP (Evans et al., 1994; Sevilla et al., 2000). Since
-DG in the spleen is expressed preferentially in the professional antigen-presenting DEC 205+ and CD11c+ DC, the result is selective infection of these cells that may alter their function and cause immunosuppression (Sevilla et al., 2000).
The present study demonstrates that high affinity binding LCMV variants like clone-13 compete successfully with laminin-1 for -DG binding and that
-DGassociated laminin-1 clusters at the cell surface did not affect clone-13 infection significantly. In contrast, the low affinity binding variant ARM53b was displaced from its receptor binding site by soluble laminin-1, and laminin-1 clusters at the cell surface reduced ARM53b infection. Based on their overlapping binding sites on
-DG (Talts et al., 1999), ECM components like laminin-1, laminin-2, perlecan, and agrin may provide a barrier for infection of cells by low affinity binding LCMV variants and influence tissue tropism and, as a consequence, the outcome of disease. Especially in organs with low concentrations of
-DG, such as the spleen (Durbeej et al., 1998),
-DGassociated ECM may be an important determinant for the anatomic distribution of LCMV variants with different receptor binding affinities. Therefore, we compared the expression pattern of laminin in the spleen with the distribution of ARM53b and clone-13. In line with previous studies (van den Berg et al., 1993), we found particularly strong laminin expression in the marginal zone at the periphery of the white pulp. Since laminin expression in the marginal zone extensively colocalized with the expression CD11c, a marker for
-DG+ DC (Sevilla et al., 2000), this area likely represents a major site of infection of these cells by LCMV. Accordingly, 23 d after i.v. inoculation, cells in the marginal zone were infected heavily with clone-13, whereas ARM53b infected cells were virtually absent from this region but localized primarily to the surrounding red pulp. The overlap between laminin expression and infection with clone-13 suggests that this virus efficiently infects cells surrounded by laminin. By contrast, the lack of association between laminin expression and the distribution of ARM53b suggests, in agreement with our in vitro studies, that laminin interferes with ARM53b infection. We speculate that high receptor binding affinity may enable variants like clone-13 to displace
-DGassociated ECM molecules from the surface of CD11c+ DCs in the marginal zone, resulting in infection of these cells. Our earlier studies showed that >80% of such DCs are infected with clone-13 (Sevilla et al., 2000). Preliminary studies showed that infected DC subsequently migrate into the white pulp and disseminate the virus into the T cell area. Low affinity binders like ARM53b that are unable to displace
-DGassociated ECM components infect small numbers of CD11c+ DC in the marginal zone with <10% of these cells infected by this virus (Sevilla et al., 2000). Because of the lower binding affinity to
-DG, resulting in less efficient infection of DCs, ARM53b may be excluded from the white pulp and is found in the red pulp. The more efficient infection of cells in the red pulp by ARM53b when compared with clone-13 may reflect the ability of ARM53b to use alternative cellular receptors for infection, a possibility that is supported by recent data from our laboratory (Smelt et al., 2001). In addition to spleen, expression of laminin is found in other lymphoid tissues that may represent sites of LCMV infection of DCs (van den Berg et al., 1993; Jaspars et al., 1995; Nilsson et al., 1998). However, the detailed distribution of laminin isoforms in these tissues is currently poorly understood. Since recent studies suggest a more restricted expression pattern of laminin-1 than previously thought (Falk et al., 1999), our staining with an antibody to laminin-1 may be due to recognition of laminin ß1 and/or
1 chains of other laminin isoforms that we are currently evaluating.
A similar in vivo selection of LCMV variants with high affinity for -DG is also observed in nonlymphoid organs, such as kidney, liver, and lung (Evans et al., 1994), suggesting that the selection of variants with high receptor binding affinity is a more general phenomenon and may represent a selective advantage during the natural infectious process.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins and antibodies
-DG was purified as described (Ervasti and Campbell, 1991). Mouse EHS laminin-1 was from GIBCO-BRL. Streptavidin coupled to agarose was from Sigma-Aldrich. Monoclonal antibodies 1-1-3 (antiLCMV-NP), WE36.1, and WE 33.1 (antiLCMV-GP1 and GP2) have been described (Buchmeier et al., 1981), as has polyclonal rabbit antibody AP83 against ß-DG (Henry and Campbell, 1998). Polyclonal rabbit antimouse EHS laminin antibody and mouse monoclonal antibody DM1A anti
-tubulin were purchased from Sigma-Aldrich, rabbit antiinfluenza HA epitope antibody Y11 from Santa Cruz Biotechnology, Inc., and hamster anti-CD11c from Serotec. FITC-conjugated goat antimouse IgG, FITC-conjugated goat antirabbit IgG, rhodamineX-labeled antiArmenian hamster IgG, and rhodamine-Xconjugated antirabbit IgG were from Jackson ImmunoResearch Laboratories. HRP-conjugated antibody against human IgGFc was purchased from Sigma-Aldrich, and HRP-conjugated antibody against mouse and rabbit IgG were from Pierce Chemical Co.
Construction of the DG deletion mutants and -DGFc fusion proteins
For the construction of the DG deletion mutants, rabbit DG cDNA fragments were amplified by PCR and inserted into the eukaryotic expression vector DGpDisplay, resulting in the following deletion mutants: DGI (H30-R168), DGD (
C182-H315), DGE (
H30-A316), DGF (
T317-P408), DGG (
G409-S485), and DGH (
H30-S485).
For the construction of the -DGFc fusion proteins, the following
-DG sequences were inserted into the expression construct IgG1Fcp-cDNA3 (Chen et al., 1996): DGFc1, amino acids 30181; DGFc2, amino acids 30316; DGFc3, amino acids 30408; DGFc4, amino acids 30485; and full-length
-DGFc, DGFc5, amino acids 30653. For detailed cloning strategies see Online supplemental material.
Production and purification of -DGFc fusion proteins
HEK293T cells were transiently transfected using calcium phosphate. 60 h after transfection, cells were lysed in 1% (wt/vol) Triton X-100, 0.1% (wt/vol) SDS, 5 mM EDTA, 1 mM PMSF in PBS with protease inhibitor complete (Roche) and lysates cleared by centrifugation (10 min, 14,000 rpm, 4°C). 20 µl/ml protein ASepharose 4B (Amersham Pharmacia Biotech) was added and incubated in the cold for 12 h. The matrix was washed twice with a 20 vol of lysis buffer, three times with a 20 vol of PBS, and then eluted with a fivefold volume 0.1 M glycine/HCl, pH 2.80. The eluate was neutralized immediately with 1 M Tris/HCl, pH 8.0, and dialyzed against PBS.
Generation of replication-deficient recombinant AdV vectors
For construction of the AdV shuttle plasmids, the cDNA fragments encoding the DG deletion mutants and wild-type DG were excised from the pDisplay-based expression constructs by HindIII and NotI and inserted into the shuttle plasmid pAdV/RSV. pAdV/RSV contains an Rous sarcoma virus long terminal repeat-driven expression cassette with a bovine growth hormone polyadenylation signal inserted between base pairs 455 and 3328 of the E1 region of human AdV5. Replication-deficient AdV vectors were generated by in vivo recombination of the AdV genomic plasmid pBHG10 with the pAdV/RSV shuttle plasmids in HEK293 cells as described (Bett et al., 1994). Twofold plaque-purified isolates were amplified, purified by CsCl gradient centrifugation, and screened for protein expression by Western blot analysis.
Virus strains, virus purification, and virus quantification
LCMV Armstrong ARM53b is a triple plaque-purified isolate of ARM CA 1371 (Dutko and Oldstone, 1983). LCMV clone-13 is a plaque-purified variant of ARM53b derived from spleen cells of an adult BALB/WEHI mouse infected persistently since birth with ARM53b (Ahmed et al., 1984). Seed stocks of all viruses were prepared by growth in BHK-21 cells. Purified virus stocks were produced and their titers determined as described (Dutko and Oldstone, 1983).
ES cells
DG-/- and DG-/+ mouse ES cells (Henry and Campbell, 1998) were maintained as described (Smelt et al., 2001). Reconstitution of susceptibility to LCMV infection by AdV-mediated gene transfer was performed as described in Cao et al. (1998). For blocking LCMV infection with soluble laminin-1, DG+/- ES cells were plated in 24-well plates coated with 5 µg/cm2 laminin-1 or fibronectin in a density of 104 cells/well. After overnight incubation, the culture medium was replaced with fresh medium containing 0, 5, 12.5, and 50 µg/ml laminin-1. 24 h later, LCMV was added at a multiplicity of infection (MOI) of 1 and incubated for 1 h at 37°C. The viral particles were removed and fresh medium containing 0, 5, 12.5, and 50 µg/ml laminin-1 added. After 16 h, infected cells were determined by immunofluorescence staining for LCMV-NP as described (Smelt et al., 2001).
Biotinylation of cell surface proteins
For cell surface biotinylation assay, DG-/- mouse ES cells (4 x 105 cells/well) were plated in gelatin-pretreated 6-well plates. After a 24-h incubation, AdV vectors were applied in an MOI of 10 and cells incubated for 40 h before cell surface labeling. Surface-specific biotinylation and isolation of biotinylated proteins was accomplished as described in Gonzalez-Dunia et al. (1997).
Immunoblotting
For isolation of total protein, cells were lysed in 1% (wt/vol) Triton X-100, 1% (wt/vol) CHAPS, 0.1% (wt/vol) SDS, 5 mM EDTA, 1mM PMSF, 1 mM iodoacetamide, 100 mM NaCl, 50 mM Tris/HCl, pH 7.5, and proteins precipitated (Wessel and Flugge, 1984). For isolation of jacalin-binding glycoproteins, cells were washed three times with PBS and solubilized in 1% (wt/vol) NP-40, 50 mM Hepes, 200 mM NaCl, 1.2 mM EDTA, 5 mM MgCl2, 5 mM CaCl2, pH 7.5, containing protease inhibitor cocktail complete and 1 mM PMSF. Lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C and incubated with 20 µl/ml jacalin conjugated to agarose (Vector Laboratories) for 12 h at 6°C. The lectin matrix was washed twice with 20 vol of solubilization buffer and three times with 50 mM Hepes, 200 mM NaCl, 0.05% (wt/vol) NP-40, 1.2 mM EDTA, 5 mM MgCl2, and 5 mM CaCl2, pH 7.5. Jacalin-bound proteins were eluted by boiling in SDS-PAGE sample buffer, separated by gel electrophoresis, and transferred to nitrocellulose. After blocking in 5% (wt/vol) skim milk/PBS, membranes were incubated with the primary antibody (AP83 antiß-DG in a dilution of 1:500; Y11 anti-HA 1:5,000; HRP-conjugated antihuman IgG1Fc, 1:20,000; DM1A anti-tubulin 1:1,000) in 2% (wt/vol) skim milk/PBS for 12 h at 6°C. HRP-conjugated secondary antibodies goat antirabbit IgG and goat antimouse IgG (Pierce Chemical Co.) were applied in a dilution of 1:5,000 for 1 h at room temperature. Blots were developed using Super Signal ECL substrate (Pierce Chemical Co.) and signals recorded on autoradiographic film (Eastman Kodak, Co.).
VOPBA, biochemical characterization of the LCMV-DG interaction, and laminin overlay assay
VOPBA was performed as described (Cao et al., 1998). The dependence of virus-DG binding on divalent cations was tested by including 1 mM CaCl2, 1 mM MgCl2, 10 mM EDTA, or 10 mM EGTA in the virus overlay solution (107 pfu/ml LCMV in VOB: 1% (wt/vol) BSA, 20 mM Hepes, 150 mM NaCl, pH 7.5). For heparin-blocking experiments, 107 pfu/ml LCMV were preincubated with 0, 25, and 100 mg/ml heparin (grade I-A from porcine intestinal mucosa [Sigma-Aldrich]) in VOB containing 5 mM CaCl2 and 5 mM MgCl2 on ice for 45 min. Sodium periodate treatment was performed on PVDF membranes by incubation with 0. 0.1, 1, and 10 mM sodium periodate in 0.1 M NaOAc, pH 5.5, for 20 min at room temperature. For neuraminidase treatment, blots were incubated with 1 U/ml neuraminidase from arthrobacter urefaciens (Calbiochem) in 0.5 M NaOAc, pH 5.5, 1% (wt/vol) NP-40, 0.1% (wt/vol) SDS for 24 h at 37°C. Blocking of virus binding by soluble sialic acid was tested by preincubation of LCMV clone-13 (107 pfu/ml) in VOB containing 1 mM CaCl2, 1 mM MgCl2, and 0, 10, and 100 mM sialic acid (Sigma-Aldrich). For blocking of virus binding by laminin-1,
-DG blots were incubated with 0, 25, and 100 µg/ml laminin-1 in laminin overlay buffer (LOB: VOB containing 1 mM CaCl2, 1 mM MgCl2) for 4 h at room temperature. 107 pfu/ml LCMV clone-13 or 5 x 108 pfu/ml LCMV ARM53b in LOB containing 0, 25, or 100 µg/ml laminin-1 were added to the blots and incubated for 12 h at 6°C. For laminin-1 displacement experiments, blots with immobilized
-DG were incubated with 107 pfu/ml LCMV clone-13 or 5 x 108 pfu/ml LCMV ARM53b in LOB for 12 h at 6°C. For displacement of bound virus, 0, 25, or 100 µg/ml laminin-1 in LOB were added and incubated at room temperature for 6 h.
Laminin overlay assay was performed as described (Durbeej and Campbell, 1999) in the presence of divalent cations or in the presence of 10 mM EDTA or 10 mM EGTA. For blocking with heparin, laminin-1 was preincubated for 1 h on ice with 0.25 and 100 mg/ml heparin in LOB.
Detection of LCMV-NP by in situ hybridization
Generation of riboprobes to LCMV-NP and in situ hybridization are described elsewhere (Sevilla et al., 2000). For the present study, C57Bl/6 mice were infected as adults (710 wk old) by i.v. inoculation of 2 x 106 pfu of LCMV ARM53b or clone-13. Spleens from three animals for each experimental group were analyzed by in situ hybridization 3 d after infection.
Immunohistochemistry
For immunofluorescence, spleens from adult uninfected C57Bl/6 mice were collected, embedded in Tissue-Tek OCT compound (Miles Diagnostic Division), and frozen on dry ice. 6 µm thick sections were cut on a cryostat, placed onto Fisher Scientific Superfrost Plus microscopic slides, dried, and fixed in cold ethanol. Rabbit antilaminin-1 antibody (1:1,000) and hamster anti-CD11c antibody (1:100) were incubated at room temperature for 1 h. As secondary antibodies, antirabbit IgG conjugated to rhodamine-X or FITC and antihamster IgG conjugated to rhodamine-X were used in a dilution of 1:100. The whole spleen composite shown in Fig. 8 a was obtained using a ZEISS Axiovert S100 microscope fitted with a 5x objective, an AxioCam digital camera, and an automated stage. Two-color registered images were captured using 490 and 570 nm excitation. For image reconstruction, MosaiX function in the KS300 image analysis software (Kontron) was used. Immunohistochemical staining using DAB as a chromogen was performed as described (Borrow et al., 1995) using rabbit antilaminin-1 in a dilution of 1:1,000 and HRP-conjugated antirabbit IgG as a secondary antibody. Sections were counter stained with Mayer's hematoxylin (Sigma-Aldrich).
Online supplemental material
Supplemental text describes the construction of the DG deletion mutants and the -DGFc fusion proteins. Fig. S1 shows in situ hybridization to compare the location of viral nucleic acids within the spleen 3 d after infection with LCMV ARM53b and clone-13, counterstained with hematoxylin and eosin. All online supplemental material available at http://www.jcb.org/content/vol155/issue2.
![]() |
Footnotes |
---|
* Abbreviations used in this paper: AdV, adenoviral; DG, dystroglycan; ECM, extracellular matrix; ES, embryonic stem; HA, hemagglutinin; LCMV, lymphocytic choriomeningitis virus, LFV, Lassa fever virus; MOI, multiplicity of infection; pfu, plaque-forming units; VOPBA, virus overlay protein binding assay.
![]() |
Acknowledgments |
---|
This work was supported by grants from the National Institutes of Health (AI45927 and AI09484 to M.B.A. Oldstone), the Human Frontier Science Program (to N. Sevilla), and the Swiss National Science Foundation (to S. Kunz). K.P. Campbell is an investigator at the Howard Hughes Medical Institute. This is publication 13957-NP from the Department of Neuropharmacology, The Scripps Institute, La Jolla, CA.
Submitted: 24 April 2001
Revised: 6 September 2001
Accepted: 10 September 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmed, R., A. Salmi, L.D. Butler, J.M. Chiller, and M.B. Oldstone. 1984. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160:521540.[Abstract]
Andac, Z., T. Sasaki, K. Mann, A. Brancaccio, R. Deutzmann, and R. Timpl. 1999. Analysis of heparin, alpha-dystroglycan and sulfatide binding to the G domain of the laminin alpha1 chain by site-directed mutagenesis. J. Mol. Biol. 287:253264.[Medline]
Bett, A.J., W. Haddara, L. Prevec, and F.L. Graham. 1994. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Natl. Acad. Sci. USA. 91:88028806.[Abstract]
Borrow, P., and M.B. Oldstone. 1992. Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus. J. Virol. 66:72707281.[Abstract]
Borrow, P., C.F. Evans, and M.B. Oldstone. 1995. Virus-induced immunosuppression: immune system-mediated destruction of virus-infected dendritic cells results in generalized immune suppression. J. Virol. 69:10591070.[Abstract]
Brancaccio, A., T. Schulthess, M. Gesemann, and J. Engel. 1997. The N-terminal region of alpha-dystroglycan is an autonomous globular domain. Eur. J. Biochem. 246:166172.[Abstract]
Buchmeier, M.J., H.A. Lewicki, O. Tonori, and M.B. Oldstone. 1981. Monoclonal antibodies to lymphocytic choriomeningitis and pichinde viruses: generation, characterization, and cross-reactivity with other arenaviruses. Virology. 113:7385.[Medline]
Cao, W., M.D. Henry, P. Borrow, H. Yamada, J.H. Elder, E.V. Ravkov, S.T. Nichol, R.W. Compans, K.P. Campbell, and M.B. Oldstone. 1998. Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science. 282:20792081.
Chen, Y., T. Maguire, and R.M. Marks. 1996. Demonstration of binding of dengue virus envelope protein to target cells. J. Virol. 70:87658772.[Abstract]
Durbeej, M., and K.P. Campbell. 1999. Biochemical characterization of the epithelial dystroglycan complex. J. Biol. Chem. 274:2660926616.
Durbeej, M., M.D. Henry, M. Ferletta, K.P. Campbell, and P. Ekblom. 1998. Distribution of dystroglycan in normal adult mouse tissues. J. Histochem. Cytochem. 46:449457.
Dutko, F.J., and M.B. Oldstone. 1983. Genomic and biological variation among commonly used lymphocytic choriomeningitis virus strains. J. Gen. Virol. 64:16891698.[Abstract]
Ervasti, J.M., and K.P. Campbell. 1991. Membrane organization of the dystrophin-glycoprotein complex. Cell. 66:11211131.[Medline]
Ervasti, J.M., and K.P. Campbell. 1993. A role for the dystrophinglycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122:809823.[Abstract]
Evans, C.F., P. Borrow, J.C. de la Torre, and M.B. Oldstone. 1994. Virus-induced immunosuppression: kinetic analysis of the selection of a mutation associated with viral persistence. J. Virol. 68:73677373.[Abstract]
Falk, M., M. Ferletta, E. Forsberg, and P. Ekblom. 1999. Restricted distribution of laminin alpha1 chain in normal adult mouse tissues. Matrix Biol. 18:557568.[Medline]
Gee, S.H., F. Montanaro, M.H. Lindenbaum, and S. Carbonetto. 1994. Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor. Cell. 77:675686.[Medline]
Gonzalez-Dunia, D., B. Cubitt, F.A. Grasser, and J.C. de la Torre. 1997. Characterization of Borna disease virus p56 protein, a surface glycoprotein involved in virus entry. J. Virol. 71:32083218.[Abstract]
Henry, M.D., and K.P. Campbell. 1998. A role for dystroglycan in basement membrane assembly. Cell. 95:859870.[Medline]
Henry, M.D., and K.P. Campbell. 1999. Dystroglycan inside and out. Curr. Opin. Cell Biol. 11:602607.[Medline]
Hohenester, E., D. Tisi, J.F. Talts, and R. Timpl. 1999. The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin. Mol. Cell. 4:783792.[Medline]
Ibraghimov-Beskrovnaya, O., J.M. Ervasti, C.J. Leveille, C.A. Slaughter, S.W. Sernett, and K.P. Campbell. 1992. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 355:696702.[Medline]
Jaspars, L.H., E. Bloemena, P. Bonnet, P. Van der Valk, and C.J. Meijer. 1995. Distribution of extracellular matrix components and their receptors in human lymphoid tissue and B-cell non-Hodgkin lymphomas. Histopathology. 26:113121.[Medline]
Kunz, S., P. Borrow, and M.B.A. Oldstone. 2001. Receptor structure and binding, cell entry. Arenavirus Biology. M.B. Oldstone, editor. Springer, Berlin, Germany. In press.
Nilsson, S.K., M.E. Debatis, M.S. Dooner, J.A. Madri, P.J. Quesenberry, and P.S. Becker. 1998. Immunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ. J. Histochem. Cytochem. 46:371377.
Oldstone, M.B. 2001. Contributions to biology and medicine from the study of the LCMV model. Arenavirus Biology. M.B. Oldstone, editor. Springer, Berlin, Germany. In press.
Oldstone, M.B., A. Tishon, J.M. Chiller, W.O. Weigle, and F.J. Dixon. 1973. Effect of chronic viral infection on the immune system. I. Comparison of the immune responsiveness of mice chronically infected with LCM virus with that of noninfected mice. J. Immunol. 110:12681278.[Medline]
Pall, E.A., K.M. Bolton, and J.M. Ervasti. 1996. Differential heparin inhibition of skeletal muscle alpha-dystroglycan binding to laminins. J. Biol. Chem. 271:38173821.
Peters, C.J., M.B. Buchmeier, P.E. Rollin, and T.G. Ksiazek. 1996. Arenaviruses. Fields Virology. B.N. Fields, D.L. Knipe, and P.M. Howley, editors. Lippincott-Raven, Philadelphia, PA. 15051519.
Rambukkana, A., H. Yamada, G. Zanazzi, T. Mathus, J.L. Salzer, P.D. Yurchenco, K.P. Campbell, and V.A. Fischetti. 1998. Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae. Science. 282:20762079.
Salvato, M., E. Shimomaye, P. Southern, and M.B. Oldstone. 1988. Virus-lymphocyte interactions. IV. Molecular characterization of LCMV Armstrong (CTL+) small genomic segment and that of its variant, Clone 13 (CTL-). Virology. 164:517522.[Medline]
Salvato, M., P. Borrow, E. Shimomaye, and M.B. Oldstone. 1991. Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence. J. Virol. 65:18631869.[Medline]
Sevilla, N., S. Kunz, A. Holz, H. Lewicki, D. Homann, H. Yamada, K.P. Campbell, J.C. de La Torre, and M.B. Oldstone. 2000. Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. J. Exp. Med. 192:12491260.
Smelt, S.C., P. Borrow, S. Kunz, W. Cao, A. Tishon, H. Lewicki, K.P. Campbell, and M.B. Oldstone. 2001. Differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics. J. Virol. 75:448457.
Southern, P.J. 1996. Arenaviridae: the viruses and their replication. Fields Virology. B.N. Fields, and P.M. Howley, editors. Lippincott-Raven, Philadelphia, PA. 15051519.
Talts, J.F., Z. Andac, W. Gohring, A. Brancaccio, and R. Timpl. 1999. Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins. EMBO J. 18:863870.
van den Berg, T.K., M. van der Ende, E.A. Dopp, G. Kraal, and C.D. Dijkstra. 1993. Localization of beta 1 integrins and their extracellular ligands in human lymphoid tissues. Am. J. Pathol. 143:10981110.[Abstract]
Wessel, D., and U.I. Flugge. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141143.[Medline]
Winder, S.J. 2001. The complexities of dystroglycan. Trends Biochem. Sci. 26:118124.[Medline]
Yamada, H., A. Chiba, T. Endo, A. Kobata, L.V. Anderson, H. Hori, H. Fukuta-Ohi, I. Kanazawa, K.P. Campbell, T. Shimizu, et al. 1996. Characterization of dystroglycan-laminin interaction in peripheral nerve. J. Neurochem. 66:15181524.[Medline]
Zinkernagel, R. 2001. Lymphocytic choriomeningitis virus. Arenavirus Biology. M.B. Oldstone, editor. Springer, Berlin, Germany. In press.