Abstract
Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.
Highlights
Ebola virus (EBOV) outbreaks continually occur and up to 90% of those infected die; currently there are no approved vaccines or antiviral therapeutics against the virus [1,2]
We further show that the enlargement of the fusion pore created by EBOV GP is unusually slow in reaching sizes necessary to pass EBOV’s genome—this is atypical of virally created fusion pores
The fraction of cells that were stained by both calcein and CMAC, 2 hr after a 10-min low pH pulse, was comparable for cell-cell fusion mediated by EBOV GP, by Jaagsiekte sheep retrovirus (JSRV) Env, and influenza A virus (IAV) hemagglutinin (HA)—all requiring low pH for fusion to proceed (Fig 1)
Summary
Ebola virus (EBOV) outbreaks continually occur and up to 90% of those infected die; currently there are no approved vaccines or antiviral therapeutics against the virus [1,2]. Endosomal interiors are difficult to control, but systems that track the entry of several other viruses into cells have been developed and employed [3,4,5,6]. These methods have relied on fusion of infectious virus or pseudovirus within cells; cell-cell fusion has not been among the systems in use for EBOV. (Some years ago there was an isolated report of EBOV GP-mediated cell-cell fusion, but this study has not been followed up by any other laboratory, including the original [8]). The system exhibits the well-known central properties of EBOV entry, providing strong support for the utility of the cell-cell fusion system to explore mechanisms of EBOV entry that are not possible or practical with whole infectious virus
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