Conservative transcription in three steps visualized in a double-stranded RNA virus

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Abstract

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8–3.5 Å) RdRp–RNA in situ structures—representing quiescent, initiation, early elongation, elongation and abortive states—under seven experimental conditions of CPV. We observed the ‘Y’-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.

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Fig. 1: CryoEM imaging and atomic modeling of de novo transcript transcription in CPV.
Fig. 2: Five functional states of CPV captured from the seven CPV samples under different conditions.
Fig. 3: Structures of RdRp and capped-terminal RNA in the quiescent and initiation states.
Fig. 4: Structures of RdRp and complete transcription bubble in the elongation state.
Fig. 5: Conformational changes of the bracelet domain and the thumb control movement of different RNA strands.
Fig. 6: Schematic illustrating state transitions and ouroboros model of de novo transcription.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. Accession codes for deposited maps in the Electron Microscopy Data Bank include those for the asymmetric reconstructed cryoEM density maps (EMD-20595 (q-CPV), EMD-20596 (S-CPV), EMD-20597 (SG-CPV), EMD-20598 (SA-CPV), EMD-20599 (SGA-CPV), EMD-20600 (SGAU-CPV) and EMD-20601 (t-CPV)) and those for the subparticle reconstructed cryoEM density maps (EMD-20581 (quiescent state), EMD-20582 (initiation state), EMD-20585 (abortive state), EMD-20586 (early-elongation state) and EMD-20587 (elongation state)). Accession codes for atomic models deposited in the Protein Data Bank include 6TY8 (quiescent state), 6TY9 (initiation state), 6TZ0 (abortive state), 6TZ1 (early-elongation state), and 6TZ2 (elongation state).

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Acknowledgements

We thank X. Yu and X. Zhang for their early efforts on this project, K. Ding and Y. He for advice in data processing and for discussion, T. Nguyen for manuscript editing, D. Weisman for illustrations, I. Atanasov for assistance in electron microscopy and P. Ge for computational support. This work was supported in part by grants from National Natural Science Foundation of China (No. 31672489 to J.S.) and the US National Institutes of Health (AI094386 and GM071940 to Z.H.Z.). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from the National Institutes of Health (1S10RR23057, 1S10OD018111 and U24GM116792) and the National Science Foundation (DMR-1548924 and DBI-1338135).

Author information

Z.H.Z. and J.S. conceived, designed and oversaw the project; J.S. and Y.Z. prepared and carried out experimental reactions; Y.C. prepared cryoEM samples, acquired cryoEM movies and performed data processing; Y.Z. built atomic models with assistance from K.Z.; Y.Z., Y.C. and Z.H.Z. interpreted the structures; Z.H.Z. and Y.Z. wrote the initial draft of the paper and all authors edited and approved the paper.

Correspondence to Jingchen Sun or Z. Hong Zhou.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Asymmetry reconstruction workflow for all CPV samples and the comparison of 10 RdRp with capped-terminal RNA in the quiescent state.

a,b, Blue arrows with explanatory text denote data-processing steps; purple text describes data properties.

Extended Data Fig. 2 Subparticle reconstruction workflows for SGA-CPV.

Subparticle reconstruction workflows for SGA-CPV. Arrows with associated text denote data-processing steps. Purple text describes the properties of the data.

Extended Data Fig. 3 Resolution verification.

a,b, Local (a) and Fourier shell correction (FSC) (b) resolution evaluation for the structures representing the five states. c,d, Density map (mesh) and atomic model (sticks) of a helix in RdRp (c) and in CSP-A (d), showing side chain densities of similar quality. e,f, RNA density map (semi-transparent grey) and atomic model (mainchains in ribbons and bases in sticks) of the capped-terminal RNA in the initiation state (e) and of the transcription bubble in the elongation state (f).

Extended Data Fig. 4 De novo transcription inside the polymerase core.

ac, Structures of RdRP (surface representation) and transcribing complex (density, tan) in initiation state (a), early-elongation state (a) and elongation state (c). df, Magnified views of the solid squared area in ac. Densities (gray) and ribbon models (color) show the details of the de novo transcription in initiation state (d), early-elongation state (e) and elongation state (f). g, Comparison of the priming loop and the switch loop in initiation state, early-elongation state and elongation state. h,i, Magnified views of the dotted square in a and b. A newly discovered Mg2+ ion binds inside a negatively charged pocket of the active site in both initiation (h) and early-elongation (i) states.

Extended Data Fig. 5 Comparisons of the tail-terminal dsRNA and the module A in all the 5 states.

ae, Models show module A (ribbon models) (aa 982–1010) and the tail-terminal dsRNA/transcription bubble (densities) in quiescent state (a), abortive state (b), initiation state (c), early-elongation state (d) and elongation state (e). fi, Comparisons of the tail-terminal dsRNA positions at the quiescent state and abortive state (f), the abortive state and initiation state (g), the initiation and early-elongation states (h) and the early-elongation and elongation states (i).

Extended Data Fig. 6 RdRp structures in the four sequential states.

ad, Surface representation models of the four determined states of RdRp: quiescent (a), initiation (b), early-elongation (c) and elongation (d). eg, Superpositions of RdRp domains in the four sequential states, comparing conformational changes between states.

Extended Data Fig. 7 Comparisons of the bracelet domain in the four sequential states.

ad, Ribbon models of the bracelet domain in the quiescent (a), initiation (b), early-elongation (c) and elongation (d) states.

Extended Data Fig. 8 Comparisons of the RdRp in the abortive state and the four sequential states.

ad, Superposition of RdRp structures in abortive state and other four sequential states, shown in full RdRp (left) and as separated domains (left).

Extended Data Fig. 9 RNA trajectories in the ouroboros model of endogenous transcription and experimental support.

a,b, Schematic illustrations showing two hypothetical trajectories of genomic RNA during transcription. If the capped-terminal RNA and the tail-terminal dsRNA that interact with the same RdRp do not belong to the same segment of genomic RNA, since the lengths of the various segments of genomic RNA are not the same, shorter RNA segments would lose their driving force, leading to an RNA ‘traffic jam’ (a). However, if the capped-terminal RNA and the tail-terminal dsRNA that interact with the same RdRp do belong to the same segment of genomic RNA, individual rounds of transcription would be independent and would not affect others’, allowing simultaneous rounds of transcription to run smoothly (b). c, Subparticle classification results supporting the ouroboros model. An example SGA-CPV cryoEM micrograph is displayed in grayscale and the identified states of sub-particles are indicated for 7 viral particles. The classification results indicate that RNA transcription by RdRp in different sub-particles within the same virion is not synchronized and could be in different states.

Supplementary information

Supplementary Information

Supplementary Results, Discussion, Methods, References and Figs. 1–3

Reporting Summary

Supplementary Table 1

Five functional states captured in the seven CPV samples.

Supplementary Video 1

Colored surface views of the asymmetric reconstruction of SGA-CPV, first showing capsid proteins (TP: blue; CSP: green), followed by genomic RNA (yellow) and TEC (RdRp: magenta; VP4: cyan), and ending with the ten TEC inside.

Supplementary Video 2

Showing the structures of RdRp and associated RNA in the initiation state in greater detail.

Supplementary Video 3

Showing in greater detail the structures of the RdRp and the complete transcription bubble in the elongation state.

Supplementary Video 4

Showing morphing of the three consecutive steps of conformational changes across the four sequential states.

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Cui, Y., Zhang, Y., Zhou, K. et al. Conservative transcription in three steps visualized in a double-stranded RNA virus. Nat Struct Mol Biol 26, 1023–1034 (2019) doi:10.1038/s41594-019-0320-0

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