Fig. S1. Inhibition of transferrin receptor endocytosis by the dominant negative Dyn mutant. HEK 293T cells were transfected as described in Fig. 1. Forty-eight hours post transfection, cell-surface TfR was labelled with specific Abs at 4°C prior to incubating cells at 37°C for the indicated time intervals to allow endocytosis. Cells were then incubated at 4°C in presence of appropriate secondary Abs and pre-coupled anti-HLA-DR Abs (L243). The graph depicts the relative levels of TfR at the cell surface of HLA-DR-positive cells (time 0 was arbitrarily set at 1.0) and represents the loss of TfR-specific signal resulting from endocytosis. These results are representative of the data obtained in three independent experiments.


Fig. S2. Plasma membrane location in the gradient, as assessed by alkaline phosphatase activity, is not affected by the expression of HLA-DR. HEK 293T cells were transfected with the HxBc2 provirus together with empty (DR-, blue line) or HLA-DR (DR+, red line) vectors. Two days after transfection cells were lysed and subcellular fractionation by optiprep gradient centrifugation performed as described in Experimental procedures. Fourteen fractions were collected from the top of the gradient and enzymatic assays were performed to identify alkaline phosphatase-containing fractions as described (Finzi et al., 2007). Briefly, 25 μl of each fraction was mixed with 15 μl of 4 mM 4-methylumbelliferyl phosphate (Sigma-Aldrich) dissolved in a citrate buffer [50 mM citric acid, 70 mM Na2HPO4 (pH 4.5)]. After 2 h of incubation at 37°C, the reaction was stopped by using 50 μl of glycine buffer [100 mM glycine (pH 10.4), 10 mM EDTA, 2% Triton X-100]. Samples were excited at 355 nm and read at 460 nm on a multiple reader (Wallac Victor, Wallac Oy, Turku, Finland).


Fig. S3. HLA-DR cell-surface expression is necessary to mediate HIV-1 internalization from the PM. HEK 293T cells were transfected with the HxBc2 provirus together with the MARCH-8-expressing construct (pCS2 MARCH-8myc6) and pBud-DR (DR+) or empty (DR-) vectors.

A. Two days after transfection, cells were metabolically labelled with [35S]Met-Cys for 10 min and chased for various times prior to cell lysis and subcellular fractionation by optiprep gradient centrifugation. The quality of the fractionation procedure was evaluated as shown in Fig. S2. Gag-related products were detected by immunoprecipitation of each fraction with monoclonal anti-p24 Abs.

B. Virus particles released during the pulse-chase analysis were pelleted, lysed and immunoprecipitated as in A.

C. Quantification of the relative amounts of mature Gag products (p25 and p24) detected in PM- and LE/MVB-associated fractions (1–6 and 13–14 respectively) after 30 min of chase. Data are representative of two independent experiments ± SD.

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