The sequential steps in the alphavirus membrane fusion pathway have been postulated based on (1) the pre- and post-fusion crystal structures of the viral fusion protein E1, (2) the protein organization of alphaviruses determined using cryo-electron microscopy (cryo-EM) image reconstruction and (3) biochemical studies. However, the molecular structures of the hypothesized fusion intermediates have remained obscure due to technical difficulties related to the dynamic nature of the fusion process. We propose to capture two pre-fusion Sindbis virus intermediates on a TEM grid by employing various types of target membranes, fusion inhibitors and functionalized TEM grids that have affinity for virus or liposomes. Our focus is on (1) the initial interaction between Sindbis virus and a target membrane at low pH and (2) the arrangement of E1 pre-fusion trimers and membrane curvature. In the long term, through the use of nanotechnology, virology methods and cryo-EM image reconstruction, we aim to characterize the conformation, oligomerization and organization of viral structural proteins in the context of membrane remodeling, curvature changes and viral infection. This work will lead to new research avenues for studying membrane fusion of enveloped viruses and serve as the basis for developing novel anti-viral strategies to combat outbreaks of disease-causing alphaviruses.
A key step in the process of host cell infection by enveloped viruses, such as influenza virus, Ebola virus, West Nile virus and Sindbis virus, is fusion of the virus envelope with the host cell membrane. With this research, we aim to elucidate the structural details of the process driven by Sindbis viral fusion proteins, and provide a general approach for structural investigation of the membrane fusion carried out by enveloped viruses. The knowledge gained from such studies will provide potential targets for anti-viral reagents.
