Tick-Borne Encephalitis Virus: A Structural View

Abstract

Tick-borne encephalitis virus (TBEV) is a growing health concern. It causes a severe disease that can lead to permanent neurological complications or death and the incidence of TBEV infections is constantly rising. Our understanding of TBEV’s structure lags behind that of other flaviviruses, but has advanced recently with the publication of a high-resolution structure of the TBEV virion. The gaps in our knowledge include: aspects of receptor binding, replication and virus assembly. Furthermore, TBEV has mostly been studied in mammalian systems, even though the virus’ interaction with its tick hosts is a central part of its life cycle. Elucidating these aspects of TBEV biology are crucial for the development of TBEV antivirals, as well as the improvement of diagnostics. In this review, we summarise the current structural knowledge on TBEV, bringing attention to the current gaps in our understanding, and propose further research that is needed to truly understand the structural-functional relationship of the virus and its hosts.

Keywords: E protein; M protein; TBEV; envelope protein; flavivirus structure; maturation; nucleocapsid; prM; tick-borne encephalitis virus; virus assembly.

Figure 1

Structure of the TBEV virion. (A) Electron cryo-micrograph of TBEV particles purified from infected cells. Smooth mature particles (black arrowheads) are presented together with immature (white arrows), partially mature (white arrowhead), and damaged (black arrows) particles. The scale bar is 100 nm. The image is courtesy of Dr. T. Füzik et al. [14] and is reproduced under a Creative Commons Attribution 4.0 International License. (B) Schematic representation of the TBEV virion. Viral genome (lilac) is encapsulated by multiple copies of the C protein (green). The nucleocapsid is surrounded by a lipid membrane (light blue), in which E and M proteins (yellow and grey, respectively) are embedded; (C) Surface representation of the TBEV virion (wwPDB: 5O6A). An icosahedral asymmetric unit is outlined in black. The three E proteins within each asymmetric unit are shown in blue, red, and yellow. Symmetry axes are indicated by the black pentagon (five-fold), the triangles (three-fold), and the ellipse (two-fold); (D) Three E-M-M-E heterotetramers on the TBEV surface. Three domains of E are highlighted in red (I), yellow (II), and blue (III), and the fusion loop is highlighted in turquoise. E protein domain IV and M protein are not visible on the virion surface.

Figure 1

Structure of the TBEV virion. (A) Electron cryo-micrograph of TBEV particles purified from infected cells. Smooth mature particles (black arrowheads) are presented together with immature (white arrows), partially mature (white arrowhead), and damaged (black arrows) particles. The scale bar is 100 nm. The image is courtesy of Dr. T. Füzik et al. [14] and is reproduced under a Creative Commons Attribution 4.0 International License. (B) Schematic representation of the TBEV virion. Viral genome (lilac) is encapsulated by multiple copies of the C protein (green). The nucleocapsid is surrounded by a lipid membrane (light blue), in which E and M proteins (yellow and grey, respectively) are embedded; (C) Surface representation of the TBEV virion (wwPDB: 5O6A). An icosahedral asymmetric unit is outlined in black. The three E proteins within each asymmetric unit are shown in blue, red, and yellow. Symmetry axes are indicated by the black pentagon (five-fold), the triangles (three-fold), and the ellipse (two-fold); (D) Three E-M-M-E heterotetramers on the TBEV surface. Three domains of E are highlighted in red (I), yellow (II), and blue (III), and the fusion loop is highlighted in turquoise. E protein domain IV and M protein are not visible on the virion surface.

Figure 2

Ribbon representation of the E and M proteins as they are found in the TBEV virion. (A) Heterotetramer of two E and two M proteins. E proteins are coloured according to domain: red (I), yellow (II), blue (III), and light blue (IV), and M proteins are shown in grey. Zoom-in caption shows the stick representation of an Asn154 glycosylation site (pink) with an N-acetylglucosamine attached (violet). (B) The structure of the E protein monomer coloured according to domains. The five helices of transmembrane domain IV are indicated. (C) The structure of the M protein monomer. The peripheral membrane helix and two transmembrane helices are indicated.

Figure 3

Comparison of the sequence and tertiary structure of flavivirus C proteins. (A) Sequence alignment of C proteins from TBEV, ZIKV and KUNV. UniProt accession numbers are shown in brackets. The alignment has been done using the Espript 3.0 web server (http://espript.ibcp.fr, [45]). White characters in red boxes highlight identical residues, red characters in blue boxes indicate residues with equivalent physico-chemical properties. Residues forming α-helices (based on the KUNV C structure) are indicated above the sequences. (B) Ribbon representation of a TBEV C dimer homology model (residues 24–96) built using the I-TASSER web server (C-score = −0.77) with the ZIKV C structure (wwPDB: 5YGH) as a template. The predicted positions of the α-helices are indicated. (C) Comparison of C-protein structures from ZIKV (green, wwPDB ID: 5YGH) and KUNV (violet, wwPDB ID: 1SFK). Positions of α-helices are indicated.

Figure 4

An overview of the TBEV life cycle. The virion interacts with a receptor on the cell surface and enters the cell via the endocytic pathway. The low pH in the late endosome triggers fusion of viral and endosomal membranes which leads to virion uncoating. Viral proteins are synthesized by the ribosomes of the rough endoplasmic reticulum (ER). Genome replication occurs in virus-induced invaginations of the endoplasmic reticulum (ER) and newly synthesized genomes are captured by C protein on the cytoplasmic side of the ER. The nucleocapsid complex acquires the structural proteins E and M and a lipid envelope by budding into the ER lumen through the membrane. The spiky immature particles are transported through the Golgi network and maturate in the acidic trans-Golgi environment after a conformational change in prM and its subsequent processing by furin. The smooth mature particles egress from the infected cell along with partially mature and immature particles. The mature and partially mature particles can start a new infection cycle but the fully immature particles are incapable of fusion and, therefore, are non-infectious.

Figure 5

Conformational rearrangement of the E protein during fusion. (A) Ribbon representation of the E-M dimer at neutral pH (wwPDB: 5O6A). Zoom in captions show pH-sensing histidines in detail. The E protein is shown in grey, the M protein is shown in light blue, the fusion loop is shown in green, and the pH-sensing histidine residues are shown in orange. The lipid bilayer is shown schematically. (B) Post-fusion E trimer conformation (wwPDB: 1URZ). The E proteins are shown in grey and fusion loops are shown in green. The lipid membrane is schematically shown. Domain IV of the E protein and protein M are not shown.

Figure 6

Schematic representation of the flavivirus polyprotein and its cleavage products. Structural proteins are shown in shades of green and non-structural proteins are shown in shades of blue, shades of violet, and in pink. Black arrows indicate viral serine protease cleavage sites, triangles indicate host signal peptidase cleavage sites, the question mark indicates the cleavage site of an unknown host protease, and the red arrow indicates a furin cleavage site. The ER membrane is shown in grey and the ER lumen and the cytoplasm are indicated.

Figure 7

Surface view of the mature and the immature DENV particles (wwPDB: 3J27 and 4B03, respectively) coloured radially according to the key. Only the prM and E models are included, the membrane and nucleocapsid inside the immature particle is not visible, leading to apparent ‘holes’ on the five-fold axis of symmetry.