Structural analysis of Ebola virus entry intermediates in vitro and in situ

Ebola virus like particles are predominantly filamentous

Ebola virus (EBOV) assembles into long filamentous virions (1-15 μm) at the plasma membrane, which upon release, enter epithelial cells by macropinocytosis. The negative single stranded RNA genome is coiled across a length of ~0.9 μm and protected by the nucleocapsid composed of the nucleoprotein (NP), VP35, VP40, and VP24 proteins. The Ebola fusion glycoprotein (GP) is proteolytically processed in the late endosome by low-pH sensitive cathepsin proteases to a 19 kDa fragment, which binds to niemann-pick-C1 receptor (NPC1). The 19 kDa fragment bound to NPC1 together with yet unknown factor(s) is able to induce membrane fusion allowing release of the genome. Both the disassembly of the filamentous virus and the mechanism underlying GP mediated membrane fusion in the endosomes are poorly understood processes and have not been structurally characterized. We will use non-infectious EBOV VLPs, which are composed of five major structural proteins (GP, NP, VP40, VP35, and VP24) and are structurally similar to EBOV to study EBOV virus entry intermediates by cryo-CLEM, cryo-FIB/SEM and cryo-ET both in vitro and in living cells.

Correlative light and electron microscopy (CLEM) of EBOV VLP entry events
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