# Molecular Organisation of Tick-Borne Encephalitis Virus
## 4 Discussion
Glycosylation of flavivirus virions plays an important role in viral particle assembly, secretion and pathogenesis, but the specific function in TBEV remains poorly understood [61; 62; 63; 64; 65; 66; 67]. The TBEV glycome has recently been characterised by mass spectrometry and contains a highly heterogeneous population of N-linked glycans at Asn154 of the E protein [50]. All glycans were reported to have a core of two GlcNac residues and a single mannose residue. Fucosylation of the second GlcNac was detected in approximately halfof the glycan species [50]. Additionally, each glycan type had three to seven additional residues linked to the core mannose [50]. We observed density corresponding to the two first GlcNac residues and the Fuc at all glycosylation sites. This difference to the glycome analysis may be due to strain variation. The glycans are highly flexible, likely to be averaged out during the reconstruction and so this probably explains why no further density was interpretable. Importantly, the metastability of TBEV fusion is partially controlled by glycosylation protecting the fusion loop, as shown in here. In conclusion, the overall molecular architecture of TBEV is controlled in the virion through protein-protein and protein-lipid interactions, and glycosylation. The role of the membrane is enhanced as a flexible scaffold that clusters the M and E heterodimers, allowing ectodomain and transmembrane interactions, and yet still promoting the metastability of the virus, allowing major conformational changes in the maturation pathway through to the fusion with the endosome in a newly infected cell. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/v14040792/s1](https://www.mdpi.com/article/10.3390/v14040792/s1)]([https://www.mdpi.com/article/10.3390/v14040792/s1](https://www.mdpi.com/article/10.3390/v14040792/s1)), Figure S1: Biochemical characterisation of purified TBEV preparation 1; Figure S2: Cryo-EM reconstruction workflow; Figure S3: Local resolution of the final map; Figure S4: Comparison of H1 densities; Table S1: Cryo-EM data collection, reconstruction, and modelling statistics; Table S2: Comparative structural analysis of the distances between the M protein membrane-proximal helices (H1) of the two M proteins at the heterotetramer interface; File S1: Structure-based sequence alignment of M proteins; File S2: Structure-based sequence alignment of E proteins; File S3: aligned TBEV M protein sequences; File S4: aligned TBEV E protein sequences; File S5: M protein sequence logo; File S6: E heterodimer interactions; File S7: E protein sequence logo; File S8: PISA analysis of the lipid pockets. Conceptualisation, L.I.A.P., S.V.B., M.A. and S.J.B. ; formal analysis, L.I.A.P., S.V.B. and M.A. ; investigation, L.I.A.P., S.V.B., M.A. and A.D.; resources, A.K.O. and S.J.B. ; data curation, L.I.A.P. ; writing--original draft preparation, L.I.A.P. and S.V.B. ; writing--review and editing, L.I.A.P., S.V.B., A.D., A.K.O., M.A. and S.J.B. ; visualisation, L.I.A.P. and S.V.B. ; supervision, M.A. and S.J.B. ; project administration, S.J.B. ; funding acquisition, A.K.O. and S.J.B. All authors have read and agreed to the published version of the manuscript. The research was funded by the Swedish Research Council, reference number 2018-05851 (to S.J.B. and A.K.O. ); the Academy of Finland, grant number 315950 (to S.J.B. ); the Sigrid Juselius Foundation, grant number 95-7202-38 (to S.J.B. ); European Union's Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreements, number 765042 (ViBRANT to S.J.B. ), and number 799929 (to M.A. ); University of Helsinki Research Council (to L.I.A.P. ); the Laboratory for Molecular Infection Medicine Sweden (MIMS) (to A.K.O.). [PERSON] and [PERSON] are fellows of the Doctoral Programme in Integrative Life Science, University of Helsinki. Not applicable. **Data Availability Statement**: The raw electron microscopy data has been deposited to Electron Microscopy Public Image Archive (EMPIAR).