Pruduction of TBEV NS5 protein for structural studies

Petra Havlíčková¹, Zdeno Gardian^{1, 2}, Paulina Duhita Anindita¹, Ivana Kutá Smatanová¹, Roman Tůma¹ and Zdeněk Franta¹

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

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

zfranta@prf.jcu.cz

Tick-borne encephalitis virus (TBEV) is a major human pathogen, transmitted by ticks from family Ixodidae [1, 2]. TBEV is an enveloped virus with a ~ 11 kb positive-sense single-strand 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 virus replication, forming replication complex [5].

Non-structural protein NS5 is a large bi-functional protein comprising of two domains connected by highly flexible 10aa linker, which is important for RdRp activity as well as for the overall shape of the protein. 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 structural studies of TBEV NS5 protein. Various constructs of TBEV NS5 protein were designed: a – full length NS5 full length including both domains, b – RdRp domain + 10aa linker, c – RdRp domain + 8aa linker, d – RdRp domain without linker, e – MTase domain. Expression and purification of individual constructs was optimized and pure samples were used for initial crystallization screening applying various commercially available crystallization screens (Molecular Dimensions, Hampton Research) as well as for cryo-EM analysis.

So far, we have obtained preliminary cryo-EM data for RdRp domain, showing typical flavivirus RdRp structure at 11 Å, that will be further refined. Needle shaped protein crystals of RdRp domain grew in several crystallization conditions. Their protein origin was verified by UVEX imaging system and these conditions will be further optimized by microseeding technique in order to grow more suitable protein crystals for X-ray diffraction analysis.

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2. Taba, P., Schmutzhard, E., Forsberg, P., Lutsar, I., Ljostad, U., Mygland, A., Levchenko, I., Strle, F. & Steiner, I.(2017). Eur. J. Neurol. 24, 1214-1261.

3. Lindenbach, B. D., Murray, C. L., Thiel, H. J. & Rice, C. M. (2013). In Fields Virology. Lippincott Williams & Wilkins, PA, USA.

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.