Crystallographic studies of TBEV NS5 RdRp domain

Petra Havlíčková¹, Paulina Duhita Anindita¹, Ivana Kutá Smatanová^1,2, Roman Tůma¹ and Zdeněk Franta¹

¹Institute of Chemistry, Faculty of Science, University of South Bohemia, Branišovská 1760, České Budějovice, Czech Republic

²Institute of Microbiology, Center for Nanobiology and Structural Biology, ASCR, Zámek 136, Nové Hrady, Czech Republic

zfranta@prf.jcu.cz

Tick-borne encephalitis virus (TBEV) is a major human pathogen, transmitted by ticks from Ixodidae family [1, 2]. TBEV is an enveloped virus with a ~ 11 kb positive-sense single-stranded RNA genome that encodes a single 375 kDa polyprotein. During the infection in the host cells, the polyprotein is cleaved by cellular and viral enzymes into three structural (capsid (C), pre-membrane (prM) and envelope (E)) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins [3]. While structural proteins are involved in the architecture of new virions [4], non-structural proteins are responsible for the viral replication machinery, forming replication complex [5]. Despite many functional studies of various TBEV NS proteins, there is actually no crystal structure of any TBEV NS proteins deposited in the Protein Data Bank.

Non-structural protein NS5 is a large bi-functional protein comprising of two domains connected by highly flexible 10aa linker. N-terminal methyltransferase (MTase) domain is involved in the capping process. C-terminal part of the protein displays RNA-dependent RNA polymerase (RdRp) activity, crucial for virus replication [6].

This project focuses on 3D structure determination of TBEV RdRp domain. Two constructs of TBEV RdRp domain with modification of the flexible linker were designed. Both constructs were cloned into pET28-SUMO expression vector and the heterologous production of recombinant enzymes was optimized using E. coli BL21-CodonPlus competent cells (Agilent). SUMO-RdRp fusion proteins were purified using IMAC. SUMO fusion partner was cleaved off and the recombinant protein was further purified via reversed IMAC. Circular dichroism analysis was used to verify the correct fold of the protein and purified RdRp was then used for initial crystallization screening applying various commercially available crystallization screens (Molecular Dimensions, Hampton Research).

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4. Fuzik, T., Formanova, P., Ruzek, D., Yoshii, K., Niedrig, M. & Plevka, P. (2018). Nat. Commun. 9, 436.

5. Mackenzie, J. (2005). Traffic. 6, 967-977.

6. Bollati, M. et al. (2009). Antiviral Res. 87, 125-148.

This research is supported by ERDF No. CZ.02.1.01/0.0/0.0/15_003/000041 and GAJU 17/2019/P.