| Summary: | The cellular response to the recombinant NS1 protein of West Nile virus (NS1<sup>WNV</sup>) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1<sup>WNV</sup>: (i) the exogenous secreted form, sNS1<sup>WNV</sup>, added to the extracellular milieu; and (ii) the endogenous NS1<sup>WNV</sup>, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1<sup>WNV</sup> varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1<sup>WNV</sup> to the cell culture medium resulted in significant remodeling of the actin filament network in Vero E6 cells. This effect was not observed in SH-SY5Y and U-87MG cells, implying that the cellular uptake of sNS1<sup>WNV</sup> and actin network remodeling were dependent on cell type. In the three cell types, NS1<sup>WNV</sup>-expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1<sup>WNV</sup> proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1<sup>WNV</sup> and the viral envelope glycoprotein E<sup>WNV</sup> were also observed in WNV-infected Vero E6 cells.
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