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.
Highlights
Zaire ebolavirus (ZEBOV, Genbank:AF086833), a member of the family Filoviridae, genus Filovirus, causes a highly fatal hemorrhagic fever in humans and non-human primates
This study defines in detail, key steps of ZEBOV cellular uptake and trafficking into cells using wild type virus as well as the host factors that are responsible for permitting virus entry into cells
Clathrin and caveolar endocytosis are not involved in ZEBOV virus entry
Summary
Zaire ebolavirus (ZEBOV, Genbank:AF086833), a member of the family Filoviridae, genus Filovirus, causes a highly fatal hemorrhagic fever in humans and non-human primates. Over the past three decades numerous human outbreaks have occurred in Central Africa involving hundreds of cases with fatality rates ranging from 50–89% [1]. Outbreaks of ZEBOV infection have been implicated in deaths of tens of thousands of gorillas, chimpanzees and duikers in Central and Western Africa posing a considerable threat to the wildlife and ecology in those areas [2]. Due to a very high case fatality rate in humans, significant transmissibility of the virus, lack of effective preventive or therapeutic measures against the disease, ZEBOV is considered a serious emerging viral pathogen. As for other members of Filoviridae, ZEBOV is morphologically distinct from other animal viruses. The approximately 19 kb single-stranded, negative-sense genomic RNA complexed with nucleocapsid, VP35, VP30 and L proteins form the nucleocapsid, while VP40 forms the matrix that underlies the viral membrane [4]
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