Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification

Abstract

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.

Fig. 1

Transmission electron microscopy of ORO in HeLa cells. ORO (arrowhead) is seen in association with clathrin-coated pits (arrow) on the surface of HeLa cells. Bar = 150 nm).

Fig. 2

Upper pannel: confocal microscopy of ORO (green) and clathrin (CLAT) heavy chain (red). Image of one representative slice of HeLa cells is shown 30 min post-ORO adsorption with XY and XZ projections of slice stacks. Colocalization of ORO and clathrin is shown in yellow/orange tones. Bar = 10 μm. Middle panel: confocal microscopy of ORO (green) and EEA1 (red). Image of one representative slice of HeLa cells is shown 40 min post-ORO adsorption with XY and XZ projections of slice stacks. The lack of yellow tones indicates absence of colocalization. Bar = 30 μm. Down panel: confocal microscopy of ORO (green) and Rab7 (red). Image of one representative slice of HeLa cells is shown 40 min post-ORO adsorption with XY and XZ projections of slice stacks. Colocalization of ORO and late endosomes is shown in yellow/orange tones (arrowheads). Bar = 10 μm.

Fig. 3

Effects of drugs on the replication of ORO: 4 μg/mL of chlorpromazine (A), 40 μg/mL of nystatin (B), 4 μg/mL of chloroquine (C) or 8 nM of bafilomycin (D) were added to HeLa monolayers, at different times relative to the infection, starting 1 h before (−1) and continuing at different times post-infection with ORO. PMA did not inhibit ORO replication (E), therefore, timing of addition assay was not carried out with this drug.