Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Tuesday Speaker Abstracts

STRUCTURAL DYNAMICS OF A NON-ENVELOPED VIRUS DURING DISASSEMBLY Milan Kumar Lokshman 1 ; Gourav Shrivastav 2 ; Kirti Suhag 1 ; Kimi Azad 1 ; Manish Agarwal 3 ; Manidipa Banerjee 1 ; 1 IIT Delhi, Kusuma School of Biological Sciences, New Delhi, India 2 IIT Delhi, Chemical Engineering, New Delhi, India 3 IIT Delhi, CSC, New Delhi, India Objective: The objective of the study is to establish the stepwise conformational alterations in the capsid of a model non-enveloped virus, Flock House Virus (FHV), during disassembly. Non enveloped icosahedral viruses have highly stable and symmetric capsids; however, dynamic structural changes are needed for release of genome during cellular entry. A molecular level understanding of the disassembly pathways can be utilized to devise methods for globally effective chemical inactivation strategies. Methods: To identify disassembly intermediates in vitro, Differential Scanning Calorimetry (DSC) and PaSTRy assay was utilized at different conditions mimicking cellular entry. Altered particles were subjected to cryoelectron microscopy and single particle analysis. Mixed populations of intermediates were sorted and subjected to icosahedral or asymmetric reconstruction. In parallel, whole capsid simulations were carried out to identify dynamic regions and hydrophobic barriers to genome release. Results: Mature and immature forms of FHV were subjected to incremental heating in DSC, which resulted in the identification of two disassembly intermediates of mature FHV. The immature particle did not undergo disassembly-related conformational changes. PaSTRy assay at low pH conditions resulted in the identification of several intermediate states, including empty capsids. Single particle reconstructions of disassembling particles indicated conformational alterations including puffing of particles triggered by movement of subunits, and major alterations at symmetry axes, particularly the 5-fold and the 2-fold axes of symmetry. Asymmetric reconstructions indicated directional genome release from the 2-fold axes of symmetry, suggesting structural differences in sequentially identical capsid proteins occupying different positions in the capsid. All atom simulations of the whole capsid supported the cryoEM studies by indicating the existence of favored pathways within the capsid for externalization of flexible components (4). Conclusion: Our studies indicate that cryoelectron microscopy and whole capsid, all-atom simulation studies can be combined to create a molecular roadmap for identifying the stages of virus disassembly.

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