Structure of severe fever with thrombocytopenia syndrome virus l protein elucidates the mechanisms of viral transcription initiation

ABSTRACT Segmented negative-sense RNA viruses (sNSRVs) encode a single-polypeptide polymerase (L protein) or a heterotrimeric polymerase complex to cannibalize host messenger RNA cap

structures serving as primers of transcription, and catalyse RNA synthesis. Here, we report the full-length structure of the severe fever with thrombocytopaenia syndrome virus (SFTSV) L

protein, as determined by cryogenic electron microscopy at 3.4 Å, leading to an atomic model harbouring three functional parts (an endonuclease, an RNA-dependent RNA polymerase and a

cap-binding domain) and two structural domains (an arm domain with a blocker motif and a carboxy-terminal lariat domain). The SFTSV L protein has a compact architecture in which its

cap-binding pocket is surprisingly occupied by an Arg finger of the blocker motif, and the endonuclease active centre faces back towards the cap-binding pocket, suggesting that domain

rearrangements are necessary to acquire the pre-initiation state of the active site. Our results provide insight into the complete architecture of sNSRV-encoded L protein and further the

understanding of sNSRV transcription initiation. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL INSIGHTS INTO THE RNA-DEPENDENT RNA POLYMERASE COMPLEXES FROM HIGHLY PATHOGENIC MARBURG AND EBOLA VIRUSES Article

Open access 31 March 2025 PRE-INITIATION AND ELONGATION STRUCTURES OF FULL-LENGTH LA CROSSE VIRUS POLYMERASE REVEAL FUNCTIONALLY IMPORTANT CONFORMATIONAL CHANGES Article Open access 17 July

2020 STRUCTURAL INSIGHTS INTO RNA POLYMERASES OF NEGATIVE-SENSE RNA VIRUSES Article 25 January 2021 DATA AVAILABILITY The cryo-EM density maps and structures were deposited in the Electron

Microscopy Data Bank and Protein Data Bank with the accession codes 0828 and 6L42, respectively. The data that support the findings of this study are available from the corresponding author

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of biomacromolecular channels. _J. Cheminformatics_ 5, 39 (2013). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the Computing and Cryo-EM platforms of the

Tsinghua University Branch of the National Center for Protein Sciences (Beijing) for cryo-EM sample preparation and screening. We acknowledge computational support from Beijing Computational

Science Research Center (CSRC) and Beijing Three Axis Space Technology Co. Ltd. This work was supported by the National Program on Key Research Project of China (grant nos. 2020YFA0707500,

2017YFC0840300, 2018YFA0507200 and 2018YFE0200402), National Natural Science Foundation of China (31971126, 31770309, 81520108019 and 31670731) and Nature Science Foundation of Hubei

Province (2019CFB790). AUTHOR INFORMATION Author notes * These authors contributed equally: Panpan Wang, Lu Liu, Aijun Liu. AUTHORS AND AFFILIATIONS * MOE Key Laboratory of Protein Science

and Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China Panpan Wang, Lu Liu, Aijun Liu, Liming Yan, Zihe Rao & Zhiyong Lou * School of

Life Sciences, Tsinghua University, Beijing, China Panpan Wang, Lu Liu, Aijun Liu, Mingxu Hu & Zihe Rao * State Key Laboratory of Medicinal Chemical Biology, College of Life Science,

Nankai University, Tianjin, China Yong He, Yu Guo & Zihe Rao * Drug Discovery Center for Infectious Disease, College of Pharmacy, Nankai University, Tianjin, China Yong He, Yu Guo, Fei

Deng & Zihe Rao * State Key Laboratory of Virology and National Virus Resource Center, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Hubei,

China Shu Shen & Fei Deng * Complex Systems Division, Beijing Computational Science Research Center, Beijing, China Haiguang Liu * Department of Biology, Southern University of Science

and Technology, Shenzhen, China Chuang Liu & Peiyi Wang * 302 Hospital, Peking University, Beijing, China Yinying Lu * National Laboratory of Macromolecules, Institute of Biophysics,

Chinese Academy of Sciences, Beijing, China Zihe Rao Authors * Panpan Wang View author publications You can also search for this author inPubMed Google Scholar * Lu Liu View author

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inPubMed Google Scholar CONTRIBUTIONS Z.L., Z.R. and F.D. conceived of the project and designed the experiments. Panpan Wang, L.L., L.Y., Y.H., S.S. and Y.G. expressed and purified the

proteins. Panpan Wang, L.L. and A.L. prepared the cryo-EM sample. Panpan Wang, A.L., M.H., H.L., C.L. and Peiyi Wang collected and processed the cryo-EM data and built the model. Panpan Wang

and Y.L. performed the surface plasmon resonance experiments. Z.L. wrote the manuscript. All authors discussed the experiments and read and approved the manuscript. CORRESPONDING AUTHORS

Correspondence to Fei Deng, Zihe Rao or Zhiyong Lou. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer

Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 CRYO-EM RECONSTRUCTION. A, Raw image of

SFTSV-L particles in vitreous ice recorded at defocus values of -1.5 to -2.5 μm. Scale bar, 100 nm. B, Power spectrum of the image shown in (A), with plot of the rotationally averaged

intensity versus resolution. White circle indicates the spatial frequency corresponding to 3.0 Å resolution. C, Representative class averages. The edge of each square is 238 Å. D, Overview

of SFTSV-L reconstruction is shown in the left panel. The close-up view of a representative region in the RdRp region is shown in the right panel. Density is shown as gray mesh; the

polypeptide of the refined model is displayed as colored sticks (carbons, green; nitrogen, blue; oxygen, red). E, Fourier shell correlation (FSC) of the final 3D reconstruction following

gold standard refinement. FSC curves are plotted before (red) and after (green) masking in addition to post-correction (blue), accounting for the effect of the mask using phase

randomization. F, Local resolution estimation was performed with Relion 2.11. G, The 3DFSC sphericity was analyzed with 3DFSC2. H, The data processing scheme used to obtain the final map.

EXTENDED DATA FIG. 2 SECONDARY STRUCTURE DIAGRAM OF SFTSV-L. Secondary structure elements along the SFTSV-L sequence are shown as cylinders and arrows for ɑ-helices and β-strands,

respectively. Domains are colored as in Fig. 1. EXTENDED DATA FIG. 3 THE LARIAT DOMAIN. A-C The lariat domain is represented as a cartoon diagram in red covered by cryo-EM density in three

views. Other domains of SFTSV-L are covered by molecular surface with the same color as in Fig. 1. D, A close-up view of the lariat domain covered by the cryo-EM density. Residues for

tracing the direction of the polypeptide are indicated by dots with labels. EXTENDED DATA FIG. 4 THE ENDON DOMAIN. A, The contacts between SFTSV-L endoN domain and other domains. The

structure of SFTSV-L is displayed with the same color scheme in Fig. 1. The endoN domain is covered by a molecular surface, and other domains are shown as cartoon diagrams. The back view and

the side view of the endoN domain are shown in the right panels. The interdomain interfaces on the endoN domain are indicated by the same color as each of the interacting domains, including

the RdRp core, the PB2-N-like domain, the arm domain and the blocker motif, the CBD domain and the lariat domain. B, The orientation of the endoN domains in SFTSV-L and LACV-L. The

structures of SFTSV-L and LACV-L 1750 are aligned according to their RdRp domains. The endoN domains of SFTSV-L and LACV-L are covered by molecular surface in forest green and smudge green,

respectively. The active residues of each endoN domain are highlighted in orange. The polypeptide of other domains in SFTSV-L was represented as cartoon diagram. The cap-binding pocket in

the CBD domain is highlighted by a red dotted circle. The distance from the cap-binding pocket to the position of the active center of LACV-L endoN domain is approximately 80 Å. EXTENDED

DATA FIG. 5 ORIENTATION OF THE CBD DOMAIN AND THE BLOCKER MOTIF. This figure is related to Fig. 2. The structures of SFTSV-L A, FluA-vRNA PA-PB1-PB2 complex B, FluB1-vRNA PA-PB1-PB2 complex

C, FluB-cRNA PA-PB1-PB2 complex D, and apo FluC PA-PB1-PB2 complex E, are aligned according to their CBD domains and are shown in the same orientation in the upper panels. Because the CBD

domains from these structures are located different site from their own RdRp cores, other domains of these structures are in significantly distinct positions. In the lower panels, the CBD

domain and its interacting fragments in these structures are shown in detail. The CBD domains are covered by molecular surface, and the other fragments are shown as cartoon diagrams. The

residues constituting the cap-binding pockets are highlighted in red and the PB2 cap-627 linker3 is in blue. Note, the orientation of the detailed illustrations for FluB1 (C), FluB (D) and

FluC (E) has a ~90° clockwise rotation for clear presentation. The residues occupy the cap-binding pockets of SFTSV-L and apo FluC PA-PB1-PB2 complex are displayed as spheres. EXTENDED DATA

FIG. 6 COMPARISON OF SUBDOMAINS IN THE RDRP REGION. The structures of the RdRp region from SFTSV-L, LACV-L and IAV PA-PB1-PB2 complex are in the same orientation and color scheme as in Fig.

4b. The PA-C-like (or PA-C) domain, the RdRp core, and the PB2-N-like (or PB2-N) domain are shown separately in the right, middle and left panels. EXTENDED DATA FIG. 7 COMPARISON OF THE

PRIMING LOOP. A-D. The subdomains of the RdRp core and the PA-C-like domain of SFTSV-L (A), LACV-L (PDB code: 5AMR) (C) and FluA PA-PB1-PB2 (PDB code: 4WSB) (D) are covered by a molecular

surface with the same color scheme as that in Fig. 4b. The priming loops in three structures are shown as white cartoons. The priming loop in SFTSV-L is enlarged and is covered with cryo-EM

density in (B). The RdRp catalytic centers of the three polymerases are highlighted by the white circles. E, Density of motif F. The RdRp motifs A, B, D, F, H of SFTSV-L are shown as cartoon

diagram with the same color scheme as that of Fig. 5a. Motif F is covered by the cryo-EM density. EXTENDED DATA FIG. 8 THE ACTIVE SITE OF THE RDRP DOMAIN. A, The details of the RdRp motifs

of SFTSV-L. This figure is generally the same as Fig. 5a, with one exception that the cation, assumed to be a magnesium ion, is shown as a red sphere. In contrast, two catalytic manganese

ions from the polio virus elongation complex structure4 are shown as black spheres. B, The bound cation and surrounding residues are covered by cryo-EM densities. The bonds formed between

SFTSV-L polypeptide and the cation are labeled with distance. C, The electrostatic surface potential of SFTSV-L RdRp tunnels. The positive surface is colored blue, the negative surface, red,

with limits ±70 kbT/ec. The regions belong to the NTP entry, template entry, template exit and product exit tunnles are indicated out by dashed lines. D, The entrance of NTP entry tunnel,

and E, the entrance of template entry tunnel. Colors are as in Fig. 4. Superposition of LACV-L 1750 (5AMR) structure shows the position of the 5' vRNA (gray cartoon) in (B). The

approximate positions of NTP and template entry tunnels are bordered by dashed lines. EXTENDED DATA FIG. 9 IMPACT OF THE ENDON DOMAIN ON THE PRODUCT EXIT TUNNEL. This figure is related to

Fig. 5b. A, Same as Fig. 5b, the tunnels for template entry, NTP entry, product exit, and template exit of SFVSV-L are indicated with violet, salmon red, orange and green labels in (A). B, A

model without the endoN domain and residues 206-217 in the linker region was generated, and its tunnels were calculated and shown as the same color scheme in (A). The bottom views of the

product exit tunnel in SFTSV-L C, or the model without the endoN domain and linker residues D, is shown. EXTENDED DATA FIG. 10 PROPOSED MODELS FOR TRANSCRIPTION INITIATION AND CO-FACTOR

BINDING. A, A proposed speculative model of SFTSV transcription initiation. SFTSV-L is schematically represented, with the cap-binding pocket (red hollow stars) in the CBD domain, the RdRp

catalytic center (deep red dots) and the active center of the endoN domain (orange stars) as marked. (1) During the early event of the cap-snatching process, the lariat domain loses its

interaction with the RdRp domain to allow the L protein to transform into a fully open state. Meanwhile, the endoN domain is pulled out from the central position of the SFTSV-L, and the arm

domain shifts to induce the removal of the Arg-finger in the block motif from the cap-binding pocket of the CBD domain. (2) After the host mRNA is captured, the endoN domain employs an ~180°

rotation along its long axis, thus causing its active center to face the nucleotide chain of host mRNA, and cleaves it by using two cations as cofactors chelated by the conserved

H...PD...D/E...K motif. If the structures of LACV-L 1750 and SFTSV-L are aligned using their RdRp cores as the reference, the cap-binding pocket in the SFTSV-L CBD domain will have a

distance of ~80 Å relative to the position of the LACV-L endoN domain active center (Supplementary Fig. 5). We reason the lack of C-terminal parts in the structure of LACV-L 1750 abolishes

the extensive interdomain interactions to maintain the compact structure and allow its endoN domain to be in a fully open state. (3) Then, the CBD anchors the snatched cap structure with

additional nucleotides and transfers the 3' end nucleotide chain to the RdRp catalytic cavity. (4) The transcription of virus mRNA is thus elongated with the guidance by vRNA as the

template, and the products will be released from the product exit tunnel. In this stage, the endoN domain is likely to stay out of the center of the L protein, allowing the product exit

tunnel to be in a fully open state. Meanwhile, the lariat motif will go back to its initial state. B, The structures of SFTSV-L and RSV-L-P complex5 are aligned by their RdRp core and shown

in the same orientation. The colors for SFTSV-L domains are the same as those in Fig. 1. The RdRp and the PRNTase domains of RSV-L are shown in pale cyan and cyan, respectively. The P

proteins bound to RSV-L is shown as cartoon diagram, and its structurally equivalent position in SFTSV-L is framed out. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Fig.

1 and Table 1. REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wang, P., Liu, L., Liu, A. _et al._ Structure of severe fever with

thrombocytopenia syndrome virus L protein elucidates the mechanisms of viral transcription initiation. _Nat Microbiol_ 5, 864–871 (2020). https://doi.org/10.1038/s41564-020-0712-2 Download

citation * Received: 25 November 2019 * Accepted: 19 March 2020 * Published: 27 April 2020 * Issue Date: 02 June 2020 * DOI: https://doi.org/10.1038/s41564-020-0712-2 SHARE THIS ARTICLE

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