Figure 3.

UUKV Entry Is Mainly Clathrin Independent

(A) UUKV-RED (moi ∼7.5) was bound to cells and then samples shifted to 37°C in the presence of NH₄Cl (50 mM) for 40 min to allow internalization. Cells were subsequently treated with TCEP (a membrane-impermeable reducing agent) to distinguish internalized from external particles and analyzed by wide-field microscopy.

(B) Internalization time course of ³⁵S-labeled UUKV (moi ∼3). The fraction that could not be removed by trypsin treatment was considered as background and subtracted from other values. Error bars indicate SD.

(C) Cells were exposed to UUKV on ice and subsequently warmed rapidly to 37°C to allow internalization. Using EM, viruses were very rarely seen in CCP and CCV but often in small, uncoated vesicles (EE #1). UUKV was also observed in larger endosomal structures (EE #2 and #3). These vacuoles were sometimes associated with electron-dense structures on their cytosolic surfaces (EE #3, white arrowhead). At later stages, viruses were often found in MVE and in structures which could correspond to LE or autophagic vesicles.

(D) Efficiency of CHC knockdown assayed by western blotting. CHC protein levels are expressed as percentages of CHC levels in cells treated with CHC siRNAs (siCHC_1 and _2) normalized to levels of β-actin and CHC in control cells treated with negative-control siRNA (siCtrl).

(E) Cells treated with CHC siRNAs were infected with UUKV or SFV. The values were normalized to the number of cells and the infection level in samples treated with negative-control siRNA (siCtrl). NI and INF for noninfected and infected cells, respectively. Error bars indicate SD.