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. 2007 Nov 28;82(3):1505–1517. doi: 10.1128/JVI.01331-07

FIG. 4.

FIG. 4.

Initial virus binding to α-DG occurs in non-raft membranes. (A) CV1 (C) and Vero E6 (V) cells express α-DG with high-affinity binding to LCMV and laminin. α-DG was isolated from CV1 and Vero E6 cells by wheat germ agglutinin lectin affinity purification. Binding to laminin was assessed by a laminin overlay assay using 10 μg/ml mouse laminin-1 and a polyclonal anti-laminin antibody, along with ECL for detection. For the virus overlay protein binding assay, blots were probed with 107 PFU/ml purified LCMV cl-13. Bound virus was detected with MAb 83.6, an HRP-conjugated secondary antibody, and ECL. (B) Blocking of LCMV cl-13 infection in CV1 and Vero E6 cells with MAb IIH6 (anti-α-DG). Cells were blocked with the indicated concentrations of MAb IIH6 or an unrelated mouse IgM (control) for 2 h at 4°C. LCMV cl-13, rVSV-LCMV GP, or VSV (200 PFU of each) was added for 45 min, and infection was assessed by the detection of LCMV NP and VSV NP with specific MAbs and fluorescein isothiocyanate-conjugated secondary antibodies in immunofluorescence. Data points represent values for triplicate samples ± standard deviations. (C) α-DG partitions into non-raft membranes. CV1 and Vero E6 cells were extracted with 1% Triton X-100 at 4°C, and samples were floated in a linear sucrose gradient. α-DG and the DRM marker caveolin-1 (Cav1) were detected in individual fractions by Western blotting with specific antibodies. DRMs, intermediate fractions, and detergent-soluble fractions are indicated. (D) Binding of LCMV to cells does not depend on membrane cholesterol. CV1 and Vero E6 cells either were treated with 10 mM MBCD for 1 h (+MBCD) or left untreated (control). Cells then were chilled on ice and incubated with purified biotinylated LCMV (100 PFU/cell). After the indicated times, unbound virus was removed, cells were washed and lysed, and LCMV GP2 was isolated by IP. Biotinylated GP2 in IPs was detected by Western blotting using HRP-conjugated streptavidin. (E) Quantification of results shown in panel D. X-ray films were analyzed by densitometry.