Structure and function of the Toscana virus cap-snatching endonuclease Rhian Jones, Sana Lessoued, Kristina Meier, Stéphanie Devignot, Sergio Barata-García, Maria Mate, Gabriel Bragagnolo, Friedemann Weber, Maria Rosenthal, Juan Reguera To cite this version: Rhian Jones, Sana Lessoued, Kristina Meier, Stéphanie Devignot, Sergio Barata-García, et al.. Struc- ture and function of the Toscana virus cap-snatching endonuclease. Nucleic Acids Research, Oxford University Press, 2019, 47, pp.10914-10930. 10.1093/nar/gkz838. hal-02611184 HAL Id: hal-02611184 https://hal-amu.archives-ouvertes.fr/hal-02611184 Submitted on 18 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License 10914–10930 Nucleic Acids Research, 2019, Vol. 47, No. 20 Published online 4 October 2019 doi: 10.1093/nar/gkz838 Structure and function of the Toscana virus cap-snatching endonuclease Rhian Jones1,†, Sana Lessoued1,†, Kristina Meier3,Stephanie´ Devignot4, Sergio Barata-Garc´ıa1, Maria Mate1, Gabriel Bragagnolo1, Friedemann Weber 4, Maria Rosenthal 3 and Juan Reguera 1,2,* 1Aix-Marseille Universite,´ CNRS, AFMB UMR 7257, 13288 Marseille, France, 2INSERM, AFMB UMR7257,13288 Marseille, France, 3Bernhard Nocht Institute for Tropical Medicine, Department of Virology, D-20359 Hamburg, Downloaded from https://academic.oup.com/nar/article-abstract/47/20/10914/5580917 by guest on 18 May 2020 Germany and 4Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, D-35392 Giessen, Germany Received March 11, 2019; Revised September 14, 2019; Editorial Decision September 17, 2019; Accepted October 01, 2019 ABSTRACT INTRODUCTION Toscana virus (TOSV) is an arthropod-borne hu- Toscana virus (TOSV) is a member of the genus phle- man pathogen responsible for seasonal outbreaks of bovirus (family Phenuiviridae,orderBunyavirales), that in- fever and meningoencephalitis in the Mediterranean cludes several arthropod-borne human pathogens such as basin. TOSV is a segmented negative-strand RNA Sandfly fever sicilian virus (SFSV), Severe fever with throm- virus (sNSV) that belongs to the genus phlebovirus bocytopenia syndrome virus (SFTSV) or Rift Valley fever virus (RVFV), that cause outbreaks in the Mediterranean (family Phenuiviridae, order Bunyavirales), encom- basin, Asia, and sub-Saharan Africa, respectively. Symp- passing other important human pathogens such as toms range from mild to severe central nervous system in- Rift Valley fever virus (RVFV). Here, we carried out fections (TOSV, SFSV) up to hemorrhagic fevers (SFTSV, a structural and functional characterization of the RV F V ) ( 1–3). The absence of efficient drugs and vaccines TOSV cap-snatching endonuclease, an N terminal for these emerging pathogens means that there is an urgent domain of the viral polymerase (L protein) that pro- need for research and development of tools for their diag- vides capped 3OH primers for transcription. We re- nostics and treatment. port TOSV endonuclease crystal structures in the Like all segmented negative-strand RNA viruses apo form, in complex with a di-ketoacid inhibitor (sNSVs), Phenuiviridae family members use a ‘cap- (DPBA) and in an intermediate state of inhibitor re- snatching’ mechanism to initiate transcription of the viral lease, showing details on substrate binding and ac- genome. By this mechanism, host cell messenger RNAs (mRNAs) are sequestered and cleaved by the EN around 11 tive site dynamics. The structure reveals substan- nucleotides downstream of the capped 5 end. The resulting tial folding rearrangements absent in previously re- small capped RNA fragments are then used for the initia- ported cap-snatching endonucleases. These include tion of viral transcription by the L protein RNA-dependent the relocation of the N terminus and the appear- RNA polymerase (RdRp) domain. Cap-snatching has been ance of new structural motifs important for transcrip- extensively structurally and biochemically characterized tion and replication. The enzyme shows high activ- for influenza virus, which replicates in the cell nucleus and ity rates comparable to other His+ cap-snatching en- has a heterotrimeric polymerase instead of the monomeric donucleases. Moreover, the activity is dependent on L protein present in bunyaviruses (4). The comparison of conserved residues involved in metal ion and sub- the structures of influenza A and B polymerases illustrated strate binding. Altogether, these results bring new the possible mechanics underlying this process; whereby light on the structure and function of cap-snatching the cap binding domain (in the PB2 subunit), captures the cellular mRNA and orientates it towards the EN domain endonucleases and pave the way for the develop- (at the N terminus of the PA subunit) for its cleavage ment of specific and broad-spectrum antivirals. before re-directing the RNA into the RdRp catalytic site (in the PB1 subunit) for priming transcription initiation (5–7). Bunyavirales carry out cap-snatching by their mul- tifunctional L protein, which harbours the EN at the N *To whom correspondence should be addressed. Tel: +33 491 825 582; Fax: +33 491 266 720; Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2019, Vol. 47, No. 20 10915 terminus. There are no L protein EN structures available fluenza virus, the enzyme lacks essential residues of the cat- for any Phenuiviridae family members, and certain lines alytic motif and consequently lacks EN activity. This sug- of evidence suggest that the cap-snatching processes may gests that THOV has possibly evolved an alternative, and differ among the sNSVs. Firstly, considering the similar still elusive, strategy to gain access to the cap structure re- architecture of sNSV polymerases reported to date (8), quired for their mRNAs (26). for arenaviruses the length of the host-derived 5 mRNA Besides the conserved residues defining their active sites, fragments (5–6 nucleotides (9–11)) is too short to allow the cap-snatching ENs of the different sNSV families have transcription priming via the mechanism proposed for no apparent sequence similarity (25). This prevents trans- influenza virus (requiring fragments of 10 to 15 nucleotides fer of the available structural information between families. (12)). This is even more remarkable for the influenza-related Consequently, there is a gap in our knowledge regarding Thogotoviruses (THOV; family Orthomyxoviridae), that structure-to-function relationships of cap-snatching ENs contains only one heterologous nucleotide in the 5 end of for many important human pathogens. This is the case for Downloaded from https://academic.oup.com/nar/article-abstract/47/20/10914/5580917 by guest on 18 May 2020 their mRNAs (13,14). Additionally, the sources of mRNA all members of the Nairoviridae and Phenuiviridae families in the nucleus (where influenza virus replicates and tran- of the order Bunyavirales. Here, we present the crystal struc- scribes) are different from those in the cytoplasm (where ture of the Phenuiviridae member TOSV cap-snatching EN bunyaviruses replicate and transcribe). In conclusion, in the apo form and in complex with the di-ketoacid in- structural and/or mechanistic variations in the influenza hibitor 2,4-dioxo-4-phenylbutanoic acid (DPBA). We char- virus cap-snatching mechanism occur among sNSVs and acterize its enzymatic activity, specificity and the impor- still need to be understood. tance of each catalytic residue in the reaction. We identify The presence of a cap-snatching EN is a common fea- new residues involved in substrate binding and investigate ture among the sNSV polymerases. The first structural and their role in catalysis. The structure reveals new features ex- functional characterization of a cap-snatching EN was re- clusive to Phenuiviridae family that we show have functional ported for influenza virus (15,16). These structures revealed implications beyond transcription. We discuss the implica- that the EN belongs to the metal ion dependent PD-D/ExK tions of these new structural features for the cap-snatching nuclease superfamily, including many different enzymes in- mechanism in the context of the full-length L proteins and volved in various aspects of DNA metabolism (e.g. EcoRV). for the evolution of the super large L proteins found in The motif includes acidic residues responsible for metal tenuiviruses and nairoviruses. ion coordination, and a lysine responsible for the stabilisa- tion and orientation of the attacking water hydroxide nu- MATERIALS AND METHODS