# Molecular Organisation of Tick-Borne Encephalitis Virus
## 1 Introduction  
Tick-borne encephalitis virus (TBEV) is a member of the _Flavivirus_ genus, and belongs in the family _Flavivriidae_[1]. The virus is predominantly transmitted to humans by ticks, and is the causative agent of tick-borne encephalitis, a severe disease that can lead to life-long neurological sequelae. The severity of the disease depends on the virus subtype. The European and Siberian subtypes typically cause milder symptoms than the Far-Eastern subtype that has a mortality rate of up to 40% [2; 3]. Although effective vaccines exist, the number of infections is on the rise, and there are no specific antivirals available [2; 3].  
The TBEV virion consists of three structural proteins, the envelope (E), membrane (M), and capsid (C) proteins, and a positive-sense single-stranded RNA genome. The genome and multiple copies of the C protein interact to form a nucleocapsid (NC). The NC is covered by the viral envelope that consists of host-derived lipids into which 180 copies of both M and E are embedded. The fundamental building block of the virion is a heterotetramer made up of two M-E dimers [4]. The heterotetramers pack tightly together to form the icosahedrally symmetric lattice of the virion surface. The E protein is the major TBEV antigen, and is responsible for receptor binding and membrane fusion [5; 6; 7; 8; 9; 10]. The Mprotein is initially synthesised as its precursor prM, which interacts with E and protects its fusion loop from premature activation. During virus maturation, prM is proteolytically cleaved, resulting in a membrane-embedded M, and a pr peptide which dissociates from the virus [11]. The crystal structure of the E protein ectodomains and a 3.9 A cryogenic electron microscopy (cryo-EM) reconstruction of the lab-adapted Hypr strain virion have previously revealed the organisation of the surface proteins, neutralisation epitopes and the amino acid residues probably involved in low pH-activated fusion [4; 12].  
Although the _Flaviirus_ genus contains multiple distinct lineages, cryo-EM studies have primarily focused on the mosquito-borne flavivirus (MBFVs), with the exception of the tick-borne TBEV and the insect-specific Binjari virus (BinJV) [4; 13; 14]. The BinJV structure illustrated differences between the lineages as the virion surface proteins are organised into heterohexameric spikes instead of heterotetramers like in other flaviviruses [15].  
Here, we present the structure of a low-passage, European TBEV subtype (Kutusalo-14) solved with cryo-EM and single-particle reconstruction to a resolution of 3.3 A. The reconstruction reveals a new M protein conformation showing a potential virion-stabilising interaction between M proteins. These interactions occur across the interface between heterotetramers. M protein interactions likely compensate for the lower number of stabilising E-E contacts at the interface compared to other flavivirus structures. The residues responsible for the M-M interaction are highly conserved across TBEV isolates, suggesting that they have an important role in TBEV virion stabilisation. We clearly resolved two flaviviral lipid factors interacting with M and E peripheral and transmembrane helices. They are in similar positions to those recently described in Zika (ZIKV), Spondweni (SPONV), Dengue (DENV), and Usuru (USUV) viruses [16; 17; 18; 19]. Furthermore, we built additional glycan residues at the E protein Asn154 glycosylation site, which has a central role in the prevention of premature fusion.