Supplemental Text S1

Supplemental text S1

Text S1. YFV-17D entry is not mediated by the clathrin-independent (CI) pathway that drive IL2R-β endocytosis

The endocytic pathway responsible for internalizing of the interleukin-2 receptor beta (IL2R-β) is another well-characterized clathrin-independent dynamin-dependent mechanism (1, 2). This endocytic route is dependent on Rac1, the serine/threonine kinases Pak1 and Pak2, as well as on the actin-related protein cortactin (3, 4). To determinate whether 17D is internalized by the same CI pathway, Hep2β cells, which stably express IL2R-β (4), were infected with 17D in the presence or absence of Rac1, Pak1 or cortactin. siRNA-mediated silencing of Rac1, Pak1 or cortactin was efficient in Hep2β cells (Fig. S3A). The number of IL2R-β and transferrin positive vesicles present in Hep2β cells depleted of CHC, APT6V1β2, Pak1, Rac1 or cortactin, was evaluated by microscopy and quantified by measuring the fluorescence intensity of endosomes per cell as previously described (4) (Fig. S3B). Cells depleted in CHC had a roughly 80% reduction in the number of transferrin positive vesicles, as compared to control cells (Fig. S3B). IL2R-β internalization was not affected by CHC silencing (Fig. S3B). As predicted, the ATP6V1β2 depletion did not reduce the endocytosis of either IL2R-β or transferrin (Fig. S3B), as these 2 proteins do not require endosomal acidification for their internalization. The depletion of the proteins specifically involved in IL2R-β endocytosis (Pak1, Rac1 and cortactin) led to an inhibition of 66%, 60% and 40%, respectively, in IL2R-β endocytosis (Fig. S3B), whilst transferrin endocytosis was barely affected by Pak1 or Rac1 depletion (Fig. S3B). Transferrin uptake was inhibited in cortactin-depleted cells to about 30% when compared to control cells (Fig. S3B), confirming the role of cortactin in both clathrin-dependent (5) and -independent pathways (6). In contrast to IL2R-β endocytosis, reduced expression of Rac-1, Pak1 or cortactin in Hep2β cells did not affect 17D infection (Fig. S3C). These results suggest that 17D does not utilize the clathrin-independent route exploited by IL2R-β for entry.

Supplemental references

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