Figure 6.

MAL is necessary for correct transport of influenza virus HA in FRT cells. (A) Wild-type FRT cells or FRT/hMAL-ΔN cells were transfected with AM or AS oligonucleotide and plated on 24-mm-diameter tissue culture inserts. After 48 h, cells were infected with influenza virus and 2.5 h later were labeled with [³⁵S]methionine/cysteine for 2 h. Apical or basolateral surface proteins were then labeled in separate culture inserts with sulfo-NHS-biotin. After cell lysis, biotinylated proteins were immunoprecipitated with streptavidin-agarose. To detect apical or basolateral surface expression of HA and E-cadherin, the immunopre-cipitates were subjected to autoradiography to detect radiolabeled HA or to immunoblot analysis, respectively. The endogenous (rMAL) or the exogenous hMAL-ΔN levels in these cells were examined by immunoblot analysis with mAb 6D9. (B and C) Quantitative analysis of the effect of MAL depletion on the polarized delivery of HA in FRT cells. The intensity of the apical and basolateral signal corresponding to radiolabeled HA from experiments in which MAL was depleted at different extensions was quantified. The values obtained are represented as percentages of radiolabeled HA on the apical or basolateral surface (B) or expressed in arbitrary units as apical and basolateral absolute surface content of radiolabeled HA (C). The results of some representative experiments are shown. The effects observed in other experiments in which rMAL was depleted to different extents (our unpublished results) were consistent with those shown. Black bars, apical; gray bars, basolateral.