# Molecular Organisation of Tick-Borne Encephalitis Virus
## 4 Discussion  
As metastable particles, TBEV virions must be both sturdy enough to protect the genome and labile enough to undergo the structural changes required for fusion. Our model allows the visualisation of key determinants of TBEV metastability including raft stabilising protein-protein and protein-lipid interactions, and fusion-modulating glycosylation. Comparison between the mosquito-borne flaviviruses, TBEV Hypr and this structure shows that extrapolation between all flaviviruses from the limited number of structures available is not always justified. The coarse generalisations hold true, but the devil is in the details. A striking difference between our reconstruction and the previous published structures is the presence of an M-M protein interaction at the inter-heterotetramer interface. The interaction contributes 12% to the total energy gain upon raft formation, improving the raft stability, compensating for the weaker E protein interactions at this interface compared to the previous structures (File S4) [4; 18; 19; 44]. The M protein interaction was not observed in the Hypr strain structure, which contains a single amino acid substitution in the M protein, Lys40 Arg [4]. In our model, Lys40 forms two key salt bridges with Glu33 of the interacting M protein. The bulkier arginine residue may not be accommodated in this position disrupting the M-M interaction. Additionally, arginine may form a competing \(\pi\)-stacking interaction with Trp37 of the same chain, resulting in a different orientation of the tryptophan residue, preventing the Trp37-Trp37 interaction [51; 52]. The formation of the M-M protein interaction at the intra-raft interface is dependent on the close proximity of the M-H1 helices in contrast to other published flavivirus structures (Table S2). During flavivirus maturation, E and M undergo a large conformational change predominantly driven by rearrangement of their membrane-associated domains [19; 53; 54; 55]. The M proteins likely come into close proximity during this process and form the observed interaction, stabilising the particle. Since the interaction interface is not observed in any mosquito-borne flaviviruses, and the interacting residues are strongly conserved only in TBEV, the M protein interface is likely unique to tick-borne flaviviruses. The two lipid densities were reasonably modelled here as PC due to the abundance of this species in flaviviruses [47]. This was possible due to the improved resolution of the membrane compared to the Hypr strain structure where the lipid density was elusive. Previously, PC 1 position was modelled as a sphingomyelin in USUV and a phosphocaramide in DENV [17; 18]. We consider both lipid classes unlikely, as phosphocaramides are exceedingly rare in flaviviruses, and our pronged density could not accommodate a single-tailed sphingomyelin molecule [47]. In the previous SPONV and USUV structures, PC 2 position was modelled as a phosphatidylethanolamine [18; 19]. However, this species usually resides on the concave leaflet of curved membranes and is uncommon in flaviviruses [56; 57]. We observe that the lipid factors stabilise the mature virion and take part in the formation of the protein raft as previously suggested [16; 17; 18; 19]. Specific deformation of the membrane curvature at these points (Figure 1) indicates that lipid rafts are probably important in assembly [58; 59]. The lipid pocket-forming residues Arg412, Trp421, His438, and Met490 of E play critical role in particle formation as shown in mutagenesis studies in ZIKV, DENV, SPONV, and TBEV [16; 17; 19; 60]. However, the functions of Thr406, Ile410 in E and Thr13 in M have not been elucidated. Since Thr406, and the presence of either an isoleucine or a leucine at site 410 are conserved throughout the flaviviruses, we propose that these residues are critical for the formation of the PC 1 pocket. In comparison, the PC 2-Thr13 interaction is only conserved in TBEV. Mutagenesis studies are required to confirm the importance of these residues in assembly.