Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Structural & Molecular Biology volume 26, pages 1023–1034 (2019)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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).

References

  1. Lawton, J. A., Estes, M. K. & Prasad, B. V. Mechanism of genome transcription in segmented dsRNA viruses. Adv. Virus Res. 55, 185–229 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shatkin, A. J. & Sipe, J. D. RNA polymerase activity in purified reoviruses. Proc. Natl Acad. Sci. USA 61, 1462–1469 (1968).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Furuichi, Y. ‘Methylation-coupled’ transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA. Nucleic Acids Res. 1, 809–822 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Farsetta, D. L., Chandran, K. & Nibert, M. L. Transcriptional activities of reovirus RNA polymerase in recoated cores. Initiation and elongation are regulated by separate mechanisms. J. Biol. Chem. 275, 39693–39701 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Borsa, J., Sargent, M. D., Lievaart, P. A. & Copps, T. P. Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process. Virology 111, 191–200 (1981).

    Article  CAS  PubMed  Google Scholar 

  6. Furuichi, Y. ‘Pretranscriptional capping’ in the biosynthesis of cytoplasmic polyhedrosis virus mRNA. Proc. Natl Acad. Sci. USA 75, 1086–1090 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mertens, P. The dsRNA viruses. Virus Res. 101, 3–13 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Zhang, X. et al. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus. Nature 527, 531–534 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, H. & Cheng, L. Cryo-EM shows the polymerase structures and a nonspooled genome within a dsRNA virus. Science 349, 1347–1350 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Ding, K., Nguyen, L. & Zhou, Z. H. In situ structures of the polymerase complex and RNA genome show how aquareovirus transcription machineries respond to uncoating. J. Virol. 92, e00774–18 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ding, K. et al. In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release. Nat. Commun. 10, 2216 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Jenni, S. et al. In situ structure of rotavirus VP1 RNA-dependent RNA polymerase. J Mol Biol 431, 3124–3138 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. He, Y. et al. In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating. Proc. Natl Acad. Sci. USA 116, 16535–16540 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhou, Z. H. in Segmented Double-Stranded RNA Viruses: Structure and Molecular Biology (ed. Patton, J. T.) Ch. 2 (Caister Academic Press, 2008).

  15. Mertens, P. P. C., Rao, S. & Zhou, Z. H. in Virus Taxonomy, VIIIth Report of the ICTV (eds Fauquet, C. M. et al.) 522–533 (Elsevier/Academic Press, 2004).

  16. Smith, R. E. & Furuichi, Y. The double-stranded RNA genome segments of cytoplasmic polyhedrosis virus are independently transcribed. J. Virol. 41, 326–329 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Furuichi, Y. & Miura, K. A blocked structure at the 5’ terminus of mRNA from cytoplasmic polyhedrosis virus. Nature 253, 374–375 (1975).

    Article  CAS  PubMed  Google Scholar 

  18. Yu, X., Jin, L. & Zhou, Z. H. 3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453, 415–419 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yu, X., Jiang, J., Sun, J. & Zhou, Z. H. A putative ATPase mediates RNA transcription and capping in a dsRNA virus. eLife 4, e07901 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Cao, G. et al. Characterization of the complete genome segments from BmCPV-SZ, a novel Bombyx mori cypovirus 1 isolate. Can. J. Microbiol. 58, 872–883 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. McClure, W. R., Cech, C. L. & Johnston, D. E. A steady state assay for the RNA polymerase initiation reaction. J. Biol. Chem 253, 8941–8948 (1978).

    Article  CAS  PubMed  Google Scholar 

  22. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Hagiwara, K., Rao, S., Scott, S. W. & Carner, G. R. Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively. J. Gen. Virol. 83, 1477–1482 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Sosunov, V. et al. Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase. EMBO J. 22, 2234–2244 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Svetlov, V. & Nudler, E. Basic mechanism of transcription by RNA polymerase II. Biochim. Biophys. Acta. 1829, 20–28 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Rothwell, P. J. & Waksman, G. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401–440 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Tsai, M. D. How DNA polymerases catalyze DNA replication, repair, and mutation. Biochemistry 53, 2749–2751 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Steitz, T. A. DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–17398 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Yang, W., Lee, J. Y. & Nowotny, M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity. Mol. Cell 22, 5–13 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Smith, R. E. & Furuichi, Y. A unique class of compound, guanosine-nucleoside tetraphosphate G(5’)pppp(5’)N, synthesized during the in vitro transcription of cytoplasmic polyhedrosis virus of Bombyx mori. Structural determination and mechanism of formation. J. Biol. Chem. 257, 485–494 (1982).

    Article  CAS  PubMed  Google Scholar 

  32. Tao, Y., Farsetta, D. L., Nibert, M. L. & Harrison, S. C. RNA synthesis in a cage-structural studies of reovirus polymerase lambda3. Cell 111, 733–745 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Wang, X. et al. Structure of RNA polymerase complex and genome within a dsRNA virus provides insights into the mechanisms of transcription and assembly. Proc. Natl Acad. Sci. USA 115, 7344–7349 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yamakawa, M., Furuichi, Y. & Shatkin, A. J. Reovirus transcriptase and capping enzymes are active in intact virions. Virology 118, 157–168 (1982).

    Article  CAS  PubMed  Google Scholar 

  35. Zarbl, H., Hastings, K. E. & Millward, S. Reovirus core particles synthesize capped oligonucleotides as a result of abortive transcription. Arch. Biochem. Biophys. 202, 348–360 (1980).

    Article  CAS  PubMed  Google Scholar 

  36. Hubin, E. A. et al. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. eLife 6, e22520 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Boyaci, H., Chen, J., Jansen, R., Darst, S. A. & Campbell, E. A. Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding. Nature 565, 382–385 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Suloway, C. et al. Automated molecular microscopy: The new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  40. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Kivioja, T., Ravantti, J., Verkhovsky, A., Ukkonen, E. & Bamford, D. Local average intensity-based method for identifying spherical particles in electron micrographs. J. Struct. Biol. 131, 126–134 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Heymann, J. B. & Belnap, D. M. Bsoft: Image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta. Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  47. Yu, I. et al. Building atomic models based on near atomic resolution cryoEM maps with existing tools. J. Struct. Biol. 204, 313–318 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta. Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Pettersen, E. F. et al. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

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

Author notes

  1. These authors contributed equally: Y. Cui, Y. Zhang.

Authors and Affiliations

  1. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Yanxiang Cui, Yinong Zhang, Kang Zhou & Z. Hong Zhou

  2. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Yinong Zhang, Jingchen Sun & Z. Hong Zhou

  3. Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

    Yinong Zhang & Jingchen Sun

Authors
  1. Yanxiang Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yinong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jingchen Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Z. Hong Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Jingchen Sun or Z. Hong Zhou.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

a–c, Structures of RdRP (surface representation) and transcribing complex (density, tan) in initiation state (a), early-elongation state (a) and elongation state (c). d–f, Magnified views of the solid squared area in a–c. 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.

a–e, 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). f–i, 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.

a–d, Surface representation models of the four determined states of RdRp: quiescent (a), initiation (b), early-elongation (c) and elongation (d). e–g, 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.

a–d, 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.

a–d, 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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41594-019-0320-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-019-0320-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing