The cellular response to the recombinant NS1 protein of West Nile virus (NS1 WNV ) 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 WNV : (i) the exogenous secreted form, sNS1 WNV , added to the extracellular milieu; and (ii) the endogenous NS1 WNV , the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1 WNV varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1 WNV 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 WNV and actin network remodeling were dependent on cell type. In the three cell types, NS1 WNV -expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1 WNV proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1 WNV and the viral envelope glycoprotein E WNV were also observed in WNV-infected Vero E6 cells.