Periodic Reporting for period 4 - BIZEB (Bio-Imaging of Zoonotic and Emerging Bunyaviruses) Reporting period: 2019-05-01 to 2020-03-31 Summary of the context and overall objectives of the project Enveloped viruses enter the cell by fusing their lipid envelope with a cellular membrane. Similar to cell–cell fusion, viral membrane fusion is mediated by fusion proteins. Several medically important viruses, notably dengue and many bunyaviruses, harbour a class II fusion protein. Atomic structures of Class II fusion proteins have been determined in pre- and post-fusion states. Furthermore, in some cases different cellular and physicochemical factors promoting fusion have been determined. However, molecular level information of different intermediate steps of the fusion process have remained uncharacterized. Our overall objective in the project was to study how the membrane fusion process happens in the presence of biological membranes at molecular level. Understanding this mechanism could contribute to developing better immunogens for vaccination and antiviral therapies. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far To address the question of how viral fusion proteins catalyze membrane fusion, we were focusing on the entry mechanism of bunyaviruses by using cutting-edge, high spatial and temporal resolution bio-imaging techniques. Bunyaviruses, and Rift Valley fever virus in particular, were chosen as a model system as they form an emerging viral threat to humans and animals, no approved vaccines or antivirals exist for human use and they were understudied compared to other class II fusion protein systems. Cryo-electron microscopy and tomography were applied to study the structures of virus and virus-like particles, in addition to virus–receptor and virus–membrane complexes. Advanced fluorescence microscopy and spectroscopy techniques were used to probe the dynamics of virus fusion membrane fusion. Our results show how the viral glycoproteins are organised on the surface Rift Valley virus. By mixing virus particles and liposomes, we were able to show how the arrangement of the fusion glycoproteins changes allowing the virus to attack the target membrane by inserting its hydrophopic fusion peptides into it. This step constitutes the first step in the membrane fusion process and proposes a step for intervention of antivirals or antibodies preventing this membrane attack from happening. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We were able to resolve, for the first time, the interaction between a viral fusion protein and a target membrane at the initiation stage. This constitutes one of the many intermediates in the viral fusion process. Mechanistic understanding of such intermediates holds potential for designing and understanding inhibitors that block the viral entry at this step. The project greatly benefited from a unique biosafety level 3 laboratory at the Oxford Particle Imaging Centre offering advanced bio-imaging techniques. The workflows established paved way for similar projects on other infectious viruses including HIV. Finally, the novel computational image processing methods developed in the project are becoming broadly applicable for the analysis of flexible biological structures, which often pose the most challenging yet interesting questions in structural biology. These methods have been made available to the scientific community following principles of open source software. In general, deciphering key steps in virus entry is expected to contribute to rational vaccine and drug design.