Avian Sarcoma Leukosis Virus and Ebolavirus Fusion Intermediates.

摘要

Enveloped viruses enter the cytosol of host cells by means of glycoproteins decorating the virion particle surface which mediate fusion between the lipid bilayer of the virus particle and the target membrane of the host cell. Using influenza virus as a model, a large body of prior work has characterized the mechanisms of class I fusion protein, but many intermediate steps along the fusion pathway remain poorly characterized. In this dissertation, we have extended assays previously utilized to study influenza virus to explore fusion intermediates of two structurally similar and atypically triggered class I fusion proteins, those of the avian retrovirus, Avian Sarcoma Leukosis Virus (ASLV), and the deadly pathogen, Ebolavirus (EBOV). In the ASLV studies, we found that fusion was biphasic with respect to pH, and that cysteine residues flanking the fusion loop were vital for the lipid-mixing stage of fusion, but not liposome binding. We also found that receptor-induced ASLV-liposome complexes were extraordinarily stable over time and amenable to visualization by cryoelectron microscopy, by which we and our collaborators directly observed rarely seen intermediates of the fusion process. In the Ebolavirus studies, we identified the protein sequence of the cathepsin-primed Ebolavirus glycoprotein (GP) intermediate and developed a liposome-binding assay to explore potential mechanisms by which fusion could be triggered. Using this assay, we showed that cathepsin-primed GP behaves like other metastable glycoproteins which can be triggered (non-physiologically) by elevated temperature or urea, but in the case of EBOV GP, this only occurs at low pH. We also provided evidence that cathepsin priming of Ebolavirus GP reduces the energy required to induce it to bind liposomes and that disulfide reduction may play a role in triggering fusion. Together, the studies comprising this dissertation provide insight into the unique fusion intermediates of the ASLV and EBOV glycoproteins and how they may relate to the general mechanism of fusion.