title: Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner.
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 caveolae-mediated 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 clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific 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.
authors.affiliation: Influenza Research Institute, Department of Pathological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Asuka
authors.initials: A
authors.lastname: Nanbo
authors.forename: Masaki
authors.initials: M
authors.lastname: Imai
authors.forename: Shinji
authors.initials: S
authors.lastname: Watanabe
authors.forename: Takeshi
authors.initials: T
authors.lastname: Noda
authors.forename: Kei
authors.initials: K
authors.lastname: Takahashi
authors.forename: Gabriele
authors.initials: G
authors.lastname: Neumann
authors.forename: Peter
authors.initials: P
authors.lastname: Halfmann
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawaoka
mesh_terms: D000818:Animals; D015153:Blotting, Western; D021941:Caveolae / Q000378:metabolism / Q000821:virology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D029043:Ebolavirus / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology; D019142:Hemorrhagic Fever, Ebola / Q000378:metabolism / Q000821:virology*; D006801:Humans; D008856:Microscopy, Fluorescence; D010873:Pinocytosis / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D058305:Sorting Nexins / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D018116:Vesiculovirus; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics; D053586:Virus Internalization; D014779:Virus Replication; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D002966:Clathrin; D012333:RNA, Messenger; D058305:Sorting Nexins; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus; D000090802:rab7 GTP-Binding Proteins; D020691:rab GTP-Binding Proteins