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Structural Insight Into Marburg Virus Nucleoprotein-RNA Complex Formation bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.452116; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Structural insight into Marburg virus nucleoprotein-RNA complex formation 2 3 Yoko Fujita-Fujiharu1,2,3, Yukihiko Sugita1,2,4, Yuki Takamatsu1,#, Kazuya Houri1,2,3, Manabu 4 Igarashi5, Yukiko Muramoto1,2,3, Masahiro Nakano1,2,3, Yugo Tsunoda1, Stephan Becker6,7, 5 Takeshi Noda1,2,3* 6 7 1 Laboratory of Ultrastructural Virology, Institute for Frontier Life and Medical Sciences, 8 Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan 9 2 Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, 53 10 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan 11 3 CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332- 12 0012, Japan 13 4 Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan. 14 5 Division of Epidemiology, International Institute for Zoonosis Control, Hokkaido University, 15 Sapporo 001-0020, Japan 16 6 Institute of Virology, University of Marburg, 35043 Marburg, Germany. 17 7 German Center for Infection Research (DZIF), Marburg-Gießen-Langen Site, University of 18 Marburg, 35043 Marburg, Germany. 19 20 # present address: Department of Virology I, National Institute of Infectious Diseases, Gakuen 21 4-7-1, Musashimurayama-city, Tokyo 208-0011, Japan 22 *correspondence to: [email protected] 23 24 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.452116; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Abstract 26 The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), 27 encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the 28 helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the 29 nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions 30 among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the 31 helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA 32 complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of 33 the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of 34 EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. 35 Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for 36 the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues 37 identified were conserved in both viruses. These findings provide a structural basis for 38 understanding the nucleocapsid formation and contribute to the development of novel antivirals 39 against MARV and EBOV. 40 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.452116; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 41 Introduction 42 Marburg virus (MARV), a close relative of the Ebola virus (EBOV), belongs to the 43 family Filoviridae and possesses a 19.1 kb non-segmented, single-stranded, negative-sense 44 RNA genome (vRNA)1. Similar to EBOV, it causes severe haemorrhagic fever in humans and 45 non-human primates with a high mortality rate, which continuously poses a threat to public 46 health. However, no vaccine or antivirals against MARV diseases have yet been licensed. The 47 nucleoprotein (NP) of MARV consisting of 695 amino acids encapsidates the vRNA to form a 48 left-handed helical complex2. The helical NP-RNA complex acts as a scaffold for the formation 49 of a nucleocapsid, which is responsible for the transcription and replication of the vRNA3,4. 50 Since the NP-RNA complex is the structural unit that constitutes the helical core structure of 51 the nucleocapsid, for which appropriate interactions between NP molecules and RNA 52 nucleotides are required, solving the NP-RNA unit structure is essential for understanding the 53 mechanisms of the nucleocapsid formation. 54 The core structure of RNA-free monomeric MARV NP, spanning residues 21 to 373, 55 features a typical bilobed fold consisting of N-terminal and C-terminal lobes, as determined 56 using X-ray crystallography at 2.9 Å resolution5, which is similar to that of the EBOV NP core 57 structure6. These two lobes are connected by a flexible hinge region and are considered to 58 clamp the RNA strand between the two lobes by electrostatic interactions. The overall three- 59 dimensional architecture of the NP-RNA complex, in which the cellular RNA was coated with 60 truncated NP comprising its N-terminal 390 residues, was visualized using cryo-electron 61 tomography (cryo-ET), showing that the helical NP-RNA complex constitutes the innermost 62 layer of the MARV nucleocapsid7. However, since the resolution was limited to the scale of a 63 few nanometres, the structural basis for the assembly of NP and RNA into the nucleocapsid 64 core largely remains unknown. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.452116; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 65 Using single-particle cryo-electron microscopy (cryo-EM), we recently determined the 66 structure of EBOV NP-RNA complex at 3.6 Å resolution8. Here, we employed the same 67 technique to determine the cryo-EM structure of the MARV NP-RNA complex. In addition, 68 we aimed to identify key residues important for NP-RNA and NP-NP interactions and viral 69 RNA synthesis using structure-based mutagenesis. 70 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.452116; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 71 Results 72 Overall structure of the MARV NP-RNA complex and its structural units 73 Similar to EBOV NP, MARV NP possesses the structural domains: an N-terminal arm, 74 an NP core composed of N- and C-terminal lobes, a disordered linker, and a C-terminal tail 75 (Fig. 1a). To determine the structure of MARV NP associated with RNA, we expressed a C- 76 terminally truncated NP which encompasses residues 1 to 395 containing the N-terminal arm 77 and the NP core domains in mammalian cells to constitute the helical complex7. Purified 78 complexes were imaged using cryo-EM (Extended Data Fig. 1a), and the structure was 79 determined by single-particle analysis at 3.1 Å resolution (Fig. 1b, Extended Data Fig. 1b-d). 80 The constituted complex shows a left-handed double helical structure (Fig. 1b, Extended Data 81 Fig. 1b), in which respective strands have similar dimensions: each strand contains single- 82 strand RNA, and 30.50 NP subunits per turn and a 128.99 Å helical pitch (rise = 4.23 Å, rotation 83 = 11.8052) with the outer and inner diameters of approximately 330 Å and 255 Å, respectively. 84 In the cryo-EM map, the density of the nucleotide bases and most of the side chains are clearly 85 visible, which enabled us to build an atomic model unambiguously showing six RNA 86 nucleotides enclosed by the N- and C-terminal lobes of NP (Fig. 1c-e). The respective 87 asymmetric NP subunits, NP-a and NP-b, show very similar conformations as shown by a 88 0.592-Å backbone RMSD (Root Mean Square Deviation) (Fig. 1c, Extended Data Fig. 1e). 89 The models of these asymmetric subunits also fit into the NP region within a low resolution 90 cryo-ET reconstruction of the MARV nucleocapsid20 (Extended Data Fig. 1f). Helix α16 91 confines the RNA strand into a cleft between the two lobes (Fig. 1d). The N-terminal arm 92 (residues 1 to 19) protrudes sideway from the N-terminal lobe and has a short 310-helix η1 (Fig. 93 1d), which is associated with a neighbouring NP molecule within the helical complex. Overall, 94 the MARV NP-RNA complex shares common structural features to that of the EBOV NP- 95 RNA unit8 (Extended Data Fig. 2), although the MARV NP-RNA complex reconstituted here bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.452116; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 96 is a double helix unlike the EBOV NP-RNA complex. Expression of the full-length wild-type 97 MARV NP also showed the formation of a double helical structure (Extended Data Fig. 3). 98 99 Amino acid residues of MARV NP involved in interactions with RNA 100 Comparison of our atomic model of the RNA-bound MARV NP with an RNA-free 101 monomeric MARV NP structure5 provides a possible mechanism of RNA encapsidation (Fig. 102 2a). A structural alignment of our RNA-bound and the previously reported RNA-free NP5 103 molecules exhibits a 0.885-Å backbone RMSD, suggesting no drastic conformational changes 104 in the overall structure. Local conformational changes are observed mainly in the C-terminal 105 lobe probably to clamp the RNA strand.
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