Structure and biological functions of TBEV Capsid protein

M. Selinger^1,2, R. Novotný^3,4, J. Sýs^3,5, J. A. Roby⁶, H. Tykalová¹, G. S. Ranjani⁷, M. Vancová², F. Kaufman³, M. E. Bloom⁸, Z. Zdráhal⁷, L. Grubhoffer^1,2, J. K. Forwood⁶, R. Hrabal⁴, M. Rumlová³ and J. Štěrba¹

¹Faculty of Science, the University of South Bohemia in České Budějovice, 370 05 České Budějovice, Czech Republic

²Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, 37005 České Budějovice, Czech Republic

³Department of Biotechnology, University of Chemistry and Technology Prague, 16628 Prague, Czech Republic

⁴Laboratory of NMR Spectroscopy, University of Chemistry and Technology Prague, 16628 Prague, Czech Republic

⁵Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, 16000 Prague, Czech Republic

⁶School of Dentistry and Medical Science, Charles Sturt University, NSW, Australia, 2678

⁷Central European Institute of Technology (CEITEC), Masaryk University, 62500 Brno, Czech Republic

⁸Biology of Vector-Borne Viruses Section, Laboratory of Virology, Rocky Mountain Laboratories, NIAID/NIH, 59840 Hamilton, MT, USA

Radim3.Novotny@gmail.com

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis [1]. The primary function of its capsid protein (TBEVC) is to recruit viral RNA and form nucleocapsid [2].

Here we show the first 3D structure of a member of tick-born flavivirus C protein, TBEVC. The structure of monomeric D16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric-interfaces were identified by hydrogen deuterium exchange mass spectrometry (Fig. 1). Although assembly of flaviviruses takes place in endoplasmic reticulum-derived vesicles [2], we observed that TBEVC protein also accumulated in nuclei and nucleoli of infected cells. Predicted bipartite nuclear localization sequence (bNLS) in the TBEVC C-terminal part was confirmed experimentally. The interface between TBEVC bNLS and import adapter protein importin-alpha was described using X-ray crystallography. Co-immunoprecipitation coupled with mass spectroscopy identification revealed 214 interaction partners of TBEV C including viral E and NS5 proteins and a wide variety of proteins involved mainly in rRNA processing and translational initiation.

Described findings may substantially help to design a targeted therapy against TBEV based on the novel functions of its capsid protein.

Figure 1: NMR structure of D16-TBEVC homodimer represented as cartoon. The monomeric protein consists of four a-helices marked as a1-a4. Generated with the PyMOL program (Schrödinger).

1. Gould EA, Solomon T: Pathogenic flaviviruses. Lancet (London, England) 2008, 371(9611):500-509.

2. Barrows NJ, Campos RK, Liao KC, Prasanth KR, Soto-Acosta R, Yeh SC, Schott-Lerner G, Pompon J, Sessions OM, Bradrick SS et al: Biochemistry and Molecular Biology of Flaviviruses. Chemical reviews 2018, 118(8):4448-4482.

We thank Z.Vavrušková (Institute of Parasitology, Biology Centre, CAS, Czech Republic) for the assistance in derivation of antibodies.

Computational resources were supplied by the project "e-Infrastruktura CZ" (e-INFRA CZ LM2018140 ) supported by the Ministry of Education, Youth and Sports of the Czech Republic.

The work was supported from European Regional Development Fund; OP RDE; Project: "ChemBioDrug" (No. CZ.02.1.01/0.0/0.0/16_019/0000729).