Technical Reference #3169

3169.

Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner Asuka Nanbo; Masaki Imai; Shinji Watanabe; Takeshi Noda; Kei Takahashi; Gabriele Neumann; Peter Halfmann; Yoshihiro Kawaoka, University of Wisconsin-Madison, PLoS Pathogens, 6(3169), (2010)
Link To Paper

Abstract:
Ebolavirus (EBOV) is an enveloped single-stranded negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolaemediated endocytosis in EBOV entry; however ebolavirus virions are long filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains therefore poorly understood. To better understand Ebolavirus entry we carried out internalization studies with fluorescently labeled biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs) both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrinor caveolae-mediated endocytosis but that they co-localized with sorting nexin (SNX) 5 a marker of macropinocytosisspecific endosomes (macropinosomes). Moreover the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo was associated with plasma membrane ruffling and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection.

Materials & Methods:
Imaging of the internalization of DiI-labeled viral particles in live cells For real-time imaging of the internalization of DiI-labeled viral particles Vero cells expressing CLCa-eGFP Cav1-eGFP eGFPSNX5 eGFP-actin or eGFP-Rab7 were cultured in 35 mm glassbottom culture dishes (MatTek corporation Ashland USA) washed in 1 ml of phenol red-free MEM (Invitrogen) containing 2% FBS and 4% BSA and incubated with DiI-labeled virions in 50 ml of the same medium on ice for 30 min. The cells were washed with the ice-cold medium and incubated for various times in a temperature-controlled chamber on the stage of a confocal laser scanning microscope (LSM510 META Carl Zeiss Oberkochen Germany); the chamber was maintained at 37uC with a humidified atmosphere of 5% CO2. Images were collected with a 40x oil objective lens (C-Apochromat NA=1.2 Carl Zeiss) and acquired by using LSM510 software (Carl Zeiss). For presentation in this manuscript all images were digitally processed with Adobe Photoshop. For co-localization analysis the images were acquired randomly the number of DiI-labeled virions that co-localized with eGFP-SNX5 or eGFP-Rab7-positive vesicles were measured in 10 individual cells (approximately 10–20 dots/cell) and the percentage of co-localization in the total DiI-virions was determined for each time point. Each experiment was performed in triplicate and the results are presented as the mean 6 standard deviation.

Microscopic Technique
confocal laser scanning microscope

Cell Type(s)
Vero cells