Oral Presentation 51st Lorne Proteins Conference 2026

Capturing viral membrane fusion by cryo-ET using an in vitro reconstitution workflow (132760)

Bronte A Johnstone 1 2 , Purba Pahari 3 , Phillip Pymm 4 5 , Wai-Hong Tham 4 5 , DIbyendu K Das 3 , Debnath Ghosal 1 2
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia
  3. Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh, India
  4. Infectious Diseases and Immune Defences Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  5. Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia

 

Host-cell entry of enveloped viruses is dependent on fusion of the viral and host-cell membranes, which enables the release of viral genome into the host-cell for replication. Membrane fusion is mediated by viral fusion glycoproteins, which undergo dramatic conformational transitions from the prefusion to postfusion state to achieve fusion. Given the dynamic, heterogenous nature of membrane fusion and the requirement for both viral and host-cell membrane, static structural biology techniques are not suitable to follow the process. Instead, cryo-electron tomography (cryo-ET) is superior and enables membrane fusion intermediates to be captured and visualised in native environments. We have developed a high-throughput cryo-ET workflow to study viral membrane fusion using an in vitro reconstitution system. This system allows for visualisation of membrane fusion between virus and host-cell membranes at subnanometre resolution without biosafety hurdles or focused ion-beam (FIB) milling. We have successfully applied this workflow to study SARS-CoV-2 membrane fusion, by combining lentiviral particles pseudotyped with SARS-CoV-2 spike glycoprotein and liposomes coated with the host-cell receptors ACE2, NRP1 and TMPRSS2. Through utilising various incubation temperatures, we have visualised fusion intermediate structures along the membrane fusion pathway by cryo-ET. This included initial host-cell attachment via receptor-receptor contact, hemi-fusion and fusion pore states, as well as post-fusion vesicles that appear to undergo multiple rounds of fusion. Furthermore, in addition to cell-surface entry, receptor-mediated endocytosis is a key mechanism for host-cell entry by SARS-CoV-2. The presence of calcium and low pH have been shown to promote membrane fusion between the virus and endosomal membrane (1,2), but the effects of these conditions have not been visualised by structural methods previously. Here, using cryo-ET we reveal that these conditions promote not only virus-host-cell fusion, but also membrane interactions between viral particles to enable viral-viral fusion. Overall, using our in vitro reconstitution system and cryo-ET workflow we have progressed our understanding of spike-mediated fusion by SARS-CoV-2, with this workflow having the potential for the study of further diverse viral fusion mechanisms.

  1. Singh P, Mukherji S, Basak S, Hoffmann M, Das DK. Dynamic Ca2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry. Cell Rep. (2022). 19;39(3):110694. DOI: 10.1016/j.celrep.2022.110694
  2. Dey S, Pahari P, Mukherjee S, Munro JB, Das DK. Conformational dynamics of SARS-CoV-2 Omicron spike trimers during fusion activation at single molecule resolution. Structure. (2024). 32(11):1910-1925.e6. doi: 10.1016/j.str.2024.09.008.