FIG 5.

The molecular composition of the cellular DG complex can affect virus uptake. (A) Schematic representation of a working model of the DG-associated sarcoglycan complex in A549 cells and skeletal muscle. (B) Reconstitution of a muscle-type sarcoglycan complex in A549 cells. A549 cells were infected with recombinant AdV5 vectors expressing α-, β-, γ-, and δ-sarcoglycan (SG) and sarcospan (SSN) (A549m) or equal amounts of AdV5 expressing GFP (A549). After 48 h, cells were subjected to cell surface biotinylation with sulfo-NHS-X-biotin (biotin) or reaction buffer only (control) as described in the legend to Fig. 3B, and expression of the indicated proteins was assessed by Western blotting as described in the legend to Fig. 3. The mildly enhanced signal for functional α-DG and the weaker band for ε-sarcoglycan were consistently observed. One representative example of three independent experiments is shown. (C) Schematic of the virus internalization assay (for details, please see text). (D) Expression of a muscle-type sarcoglycan complement in A549m cells affects viral uptake. A549m and A549 cells were chilled on ice and incubated with biotin-S-S-labeled rLCMV-LASVGP (50 particles/cell) for 1 h in the cold. Unbound virus was removed, and cells were shifted to 37°C. After the indicated time points, cells were chilled on ice and treated with TCEP (+TCEP) or reaction buffer only (−TCEP). After quenching of residual TCEP, cells were lysed, viral GP was isolated by IP with MAb 83.6 to GP2. Biotinylated GP2 was detected with streptavidin-HRP in Western blots under nonreducing conditions using enhanced chemiluminescence (ECL). The top blot (+TCEP) was exposed for 5 min, and the bottom blot (−TCEP) was exposed for 30 s. One representative example of three independent experiments is shown. (E) Quantification of the three independent experiments (D). The intensity of GP2 signals in the presence or absence of TCEP was assessed by densitometry, followed by calculation of the signal ratios. For normalization, the value for the 15-min time point for A549 control cells was set at 1. Data are means ± SD (n = 3). (F) Infection of A549m and A549 cells. A549m and A549 cells in panel B were incubated with 300 PFU/well rLCMV-LASVGP (LASV) and rLCMV-VSVG (VSV), and infection was detected as described in the legend to Fig. 1D. Data are means ± SD (n = 3). One representative example of three independent experiments is shown.