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Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.

ABSTRACT: Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.

SUBMITTER: Saeed MF

PROVIDER: S-EPMC2940741 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.

Saeed Mohammad F MF Kolokoltsov Andrey A AA Albrecht Thomas T Davey Robert A RA

PLoS pathogens 20100916 9

Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outc ...[more]

PMID: 20862315

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Project description:Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.

Dataset Information

Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.

Publications

Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.

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