Human parainfluenza virus fusion complexglycoproteins imaged in action

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Project Summary Infection by human parainfluenza viruses (HPIVs) causes important lower respiratory diseases including croup, bronchiolitis and pneumonia, that lead to illness or death in millions of infants and young children worldwide. There are no vaccines or effective treatments. During viral entry, the first step of infection, the viral fusion complex – comprised of the surface glycoproteins HN (receptor-binding protein; hemagglutinin-neuraminidase) and F (fusion protein) – mediates fusion upon receptor binding. HN triggers F to undergo conformational rearrangements that promote viral entry, and these key steps are not understood. The overall goal of this proposal is to capture serial intermediate states of the HPIV3 HN-F fusion complex in native state during activation and examine the fusion mechanism using cryo-electron tomography (ET). Cryo-ET will provide direct structural evidence related to our proposed mechanisms of action of the glycoprotein complex during viral entry. Aim 1 will focus on HN’s protective role in preventing premature activation of F-mediated fusion, using cryo-ET to compare HN-F specific complexes (1.1) and small molecules that interact with HN and induce it to activate F (1.2). In aim 2, we will capture and visualize the structural rearrangements in the HN-F fusion complex during serial stages of the fusion process. Execution of these aims along with the training as outlined will prepare me to advance my field in a fashion that applies structural biology methods to important mechanistic questions in virology. I expect to characterize in unprecedented detail how HN triggers F-mediated fusion for HPIV3 and provide clear answers about the process of viral fusion that have been central and puzzling – while at the same time leading to development of cutting-edge methods in cryo-ET. Using this approach, it will be possible not only to test hypotheses that attempt to simplify the nature of the HN-F relationship, but to examine the fusion complex at specific intermediate points in time in native, authentic configurations as they exist on the virus. Our experiments will explain how the necessary intermediates form, and the mechanistic data gleaned from our results can then guide antiviral strategies, as this understanding is a prerequisite for developing ways to disable the fusion/entry phase of infection. The project is innovative in both technical and conceptual aspects and thus provides an ideal training vehicle for me to develop into a rigorous and creative independent scientist.